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346 questions with answers in MOLECULAR MICROBIOLOGY |...

产品文献产品Rainbow AgarMitoPlateSherlock SoftwareFF(丝状真菌)软件解决方案表型微阵列定制表型微阵列EcoPlate微生物鉴定微孔板实验室服务YT MicroPlateMicroStationOdin应用教育发酵生物工艺可持续农业环境监测基础研究资源类型宣传册应用说明使用说明测试面板用户指南数据库分析证书Safe ty 数据表一般过滤器:EcoPlateSafety 数据表全部清除没有可显示的帖子。\"\"LinkedInTwitterFacebook

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产品产品实验室服务应用资源网络研讨会新闻与活动贸易展览新闻发布​​公司关于我们联系方式提交样品产品文献产品Rainbow AgarMitoPlateSherlock SoftwareFF(丝状真菌)软件解决方案表型微阵列定制表型微阵列EcoPlate微生物鉴定微孔板实验室服务YT MicroPlateMicroStationOdin应用教育发酵生物工艺可持续农业环境监测基础研究资源类型宣传册应用说明使用说明测试面板用户指南数据库分析证书Safe ty 数据表一般过滤器:定制表型微阵列安全数据表全部清除没有可显示的帖子。\"\"LinkedInTwitterFacebook

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产品产品实验室服务应用资源网络研讨会新闻与活动贸易展览新闻稿公司关于我们联系我们提交样品产品文献产品Rainbow AgarMitoPlateSherlock SoftwareFF(丝状真菌)软件解决方案表型微阵列定制表型微阵列EcoPlate微生物鉴定微孔板实验室服务YT MicroPlateMicroStationOdin应用教育发酵生物工艺可持续农业环境监测基础研究资源类型宣传册应用说明使用说明测试面板用户指南数据库分析证书Safe ty 数据表常规过滤器:表型微阵列安全数据表全部清除没有可显示的帖子。\"\"LinkedInTwitterFacebook

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产品产品实验室服务应用资源网络研讨会新闻与活动贸易展览新闻发布​​公司关于我们联系方式提交样本莱斯产品文献产品Rainbow AgarMitoPlateSherlock SoftwareFF(丝状真菌)软件解决方案表型微阵列定制表型微阵列EcoPlate微生物鉴定微孔板实验室服务YT MicroPlateMicroStationOdin应用教育发酵生物工艺可持续农业环境监测基础研究资源类型宣传册应用说明使用说明测试面板用户指南数据库分析证书Safe ty 数据表一般过滤器:软件解决方案安全数据表全部清除没有可显示的帖子。\"\"LinkedInTwitterFacebook

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产品产品实验室服务应用资源网络研讨会新闻与活动贸易展览新闻发布​​公司关于我们联系方式提交样品产品文献产品Rainbow AgarMitoPlateSherlock SoftwareFF(丝状真菌)软件解决方案表型微阵列定制表型微阵列EcoPlate微生物鉴定微孔板实验室服务YT MicroPlateMicroStationOdin应用教育发酵生物工艺可持续农业环境监测基础研究资源类型宣传册应用说明使用说明测试面板用户指南数据库分析证书Safe ty 数据表一般过滤器:FF(丝状真菌)安全数据表全部清除没有帖子可显示。\"\" LinkedInTwitterFacebook

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产品产品实验室服务应用资源网络研讨会新闻与活动贸易展览新闻发布​​公司关于我们联系方式提交 Sam普莱斯Science topics: Biological ScienceMicrobiologyMolecular MicrobiologyScience topicMolecular Microbiology - Science topicExplore the latest questions and answers in Molecular Microbiology, and find Molecular Microbiology experts.Questions (346)Publications (249,533)Questions related to Molecular Microbiology1234 Mercedes Vazquezasked a question related to Molecular MicrobiologyAny suggestions on my problem with electrotransformation of pE-FLP (flippase expression vector)?Question6 answersMay 21, 2015Hi, I m trying to disrupt some genes of the genome of E. coli. For achieving this, I m following the protocol of the P1 phage in which this phage transfers genetic material from one strain to another. The recipient strain is from the keio collection, so I don t have any problem with the selection of the colonies which were succesfully trasduced.But after the confirmation of the disruption with the antibiotic screening and PCR, I tried to remove the Kn resistance using flippase. The flippase is a recombinase that recognize FRT (Flippase recognition target) sites and removes all the flanked area.This plasmid must be electroporated and then grow it at 30°C, because it has a temperature sensitive ori.I have done all of this but I have not obtained any transformant. Do you have any clue of why I don t get colonies? Can you give me some tips?Relevant answerOsama KamalJun 29, 2021AnswerMercedes Vazquez Can I as you what plasmid you are talking about? Is it pCP20?Many thanks?View0 Recommendations Abhishek Dubeyasked a question related to Molecular MicrobiologyRescue Cloning of Bacterial Genomic DNA using the EZ::TN™ R6Kγori /KAN-2 Tnp Transposome ?Question5 answersSep 28, 2015Hello everyone,I have been trying rescue cloning experiments as per the protocol suggested by http://www.epibio.com/docs/default-source/protocols/ez-tn5-lt-r6k%CE%B3ori-kan-2-gt-tnp-transposome-kit.pdf?sfvrsn=8but inspite of trying multiple times I am not able to get any colony on the plate.Can anyone help me with this ?Relevant answerTaylor M. HarrisMar 23, 2021AnswerHi Abhishek,Did you get it to work? I am using the transposome kit and the pir-116 cells now. I had no luck so far, but the control DNA works in the cells though. Do you have any recommendations?View0 Recommendations Julian Trouillonasked a question related to Molecular MicrobiologyWhich tool for operon prediction in bacterial genomes ?Question8 answersOct 8, 2020Hi,I want to predict operons on entire bacterial genomes (already annotated). I used to use operon-mapper ( https://academic.oup.com/bioinformatics/article/34/23/4118/5040321), which is great but it has been under maintenance for weeks now and I really need one now.MicrobesOnline doesn t support private genome hosting anymore and I think that the DOOR website is now down. If you know any other tool, online or not, that would allow the genome-wide prediction of operons in complete, annotated bacterial genomes, that would be of great help to me.ThanksJulianRelevant answerVincent AppiahDec 23, 2020AnswerYou can try annotating your genome with tools like PROKKA, PGAP or RAST. And look at the predicted genes and other featuresView0 Recommendations Priyanka Mishraasked a question related to Molecular MicrobiologyWhich bacteria can grow on 10x PBS buffer?Question7 answersNov 5, 2020I have prepared PBS buffer and components for M9 medium and I store it in freeze, I do not use it if it is more than 2 weeks old. From time to time I check the sterility of these solutions and plate these solutions on N agar plate, also with a control plate. This is third time I saw something growing on PBS buffer, it was just autoclaved 3 days ago. Does somebody have any idea, which bacteria can grow on 10x PBS bufferRelevant answerDanvir RamesarNov 7, 2020AnswerWhat ive seen is that in rare cases after an autoclaved high conc PBS (phosphate-buffered saline) solution has been stored cold, it can result in precipitation which may create the impression of contamination.Cold stored PBS ( ~ 4C // -20C) should not accommodate general lab microbial contaminants to grow. Firstly, essential C and N sources are lacking and the potential of psychrophilic microbes being present under general lab conditions is highly unlikely. It s more likely that the contaminant got into your Soln when it was being carried or handled or during the frequent contamination checks and remained stationary. PBS is stable indefinitely, I have a100 mM stock for about 14 months at 4C and it was sterile, no precipitation. Don t lose sleep over it. A way to prevent this from happening in the future is to make small aliquots of PBS that are enough for your general use, completely cover them in a single layer of foil and autoclave. I am in a Bacillus lab, and the likelihood of spores being present on surfaces is quite high. Thus far i have had no contamination issues even with my nutrient-rich buffered media.Spray the foil (if you really worried about contamination, first with 10% bleach ) and then with 70% EtOH the foil and remove it before you want to use your PBS and it should be stable and sterile. Bleach and EtOH are not sporicidal w.r.t. Bacillus but for general microbes, it should be okay.Try it out and let RG comments know ?View14 Recommendations Umapriyatharshini Rajagopalanasked a question related to Molecular MicrobiologyCan anybody suggest an effective media to culture Saccharomyces pastorianus?Question7 answersMar 27, 2013I try to optimize the conditions for both liquid cultures (in Bio reactors) and agar cultures.Relevant answerRosa Garcia SanchezOct 24, 2020AnswerHello,If you want to grow them in rich media, YPD or synthetic wort media work fine. If you are after some physiological characterization in bioreactors where you would like to do carbon balances or other metabolic analysis, then you might go for a more defined media like YNB with maltose or glucose as carbon sources.With regards,RosaView0 Recommendations Seana ni Fhearghailasked a question related to Molecular MicrobiologyIsolation of bacterial DNA from plant root samples? Question4 answersSep 22, 2020As part of a research project i have obtained plant root samples that are currently frozen. I need to isolate bacterial DNA from the plant root, however i first need to remove the possible bacterial biofilm from the plant root as the bacterial DNA is of interest, not plant root DNA. Any advice would be greatly appreciated Relevant answerSalma MukhtarSep 23, 2020AnswerI agree with Debapriya Sarkar in this question but you can also use some commercially available kits, e.g., FastPrep DNA isolation kit for bacteriaView5 Recommendations Natakorn Nokchanasked a question related to Molecular MicrobiologyCan anyone suggest me how to grow Neisseria gonorrhoeae in liquid culture for DNA extraction?Question6 answersJan 30, 2017I try to use the isolated colonies of Neisseria gonorrhoeae in chocolate agar for DNA extraction, but due to their clumping when I put them into liquid to make them dissolve, it perturb me for extracting DNA in a large collection of sample.So how about the easiest way to doing that? Can I use the LB broth instead of fastidious or GCBL broth? What s the condition? Need I grow them in 5% CO2?Thanks for your suggestion.Relevant answerLoree Anne de GuzmanMay 4, 2020AnswerFB was prepared as described previously by Cartwright et al. (3), and consisted of 35 g of Columbia broth base, 5 g of glucose, 5 g of yeast extract, 2 g of neopeptone, and 0.75 g of agarose dissolved in 960 ml of distilled water. A total of 30 ml of hematin solution (0.05% [wt/vol] in 0.1 M NaOH) and 5 ml of Tween 80 (10% [vol/vol]) was then added, and the resultant broth was sterilized by autoclaving, after which 6 ml of pyridoxal solution (0.1% [wt/vol]) and 1.5 ml of NAD solution (1% [wt/vol[) were added.for mo infoArticle Cultivation of Neisseria gonorrhoeae in Liquid Media and Det... View0 Recommendations Ioanna Eleftheriadouasked a question related to Molecular MicrobiologyHow can i dilute carvacrol (oil) ?Question9 answersAug 10, 2016I have read in some papers that carvacrol can be diluted in Tween 80 (1%) and distilled water. I have tried this but carvacrol wasnt fully dissolved. After that i tried to dissolve carvacrol in Tween 80, water and ethanol. Is that right? Has anyone done this? I want  to use this mixture for epicutaneous administration in mice.Relevant answerSwati KunduApr 26, 2020AnswerFor both, in vitro in vivo study, I have dissolved carvacrol in DMSO.View4 Recommendations Asif Shahriarasked a question related to Molecular MicrobiologyHow to design multi Epitopes Vaccine Prediction against Novel Coronavirus 2019 (COVID-19) Using Immunoinformatics tools.?Question12 answersFeb 22, 2020 Severe acute respiratory syndrome coronavirus 2, formerly known as the 2019 novel coronavirus, is a positive-sense single-stranded RNA virus. It is contagious among humans and is the cause of coronavirus disease 2019. I am trying design a model of multi epitope subunit based vaccine by immunoinformatics approaches.Relevant answerTimothy OmaraApr 6, 2020AnswerFollowingView36 Recommendations Tali Langasked a question related to Molecular MicrobiologyHow do I treat BMDM or iBMM with flagellin?Question2 answersJun 8, 2015I am wanting to treat BMDM or iBMM with commercial purified Flagellin. I have looked at papers and it seems to get a decent response you have to transfect it in. Does anyone have protocols for transfection into primary cells and concentrations, time etc?Also has anyone just put purified flagellin onto cells and seen a decent IL-1b induction via ELISARelevant answerBinita Roy NandiMar 23, 2020AnswerHey, I am also trying Flagellin induction, but am unsuccessful till now.. Just to ask, did it work for you? If yes, then how?View0 Recommendations Asif Shahriarasked a question related to Molecular MicrobiologyI want to disrupt biofilm by using an electric current, what equipment can I use to achieve this?Question4 answersJan 19, 2020 I m not sure how to achieve this in a microtitre plate or petri dish, I did think by electrophoresis, but is there anything else I can use and voltage? Relevant answerKaren A. DarbinyanJan 21, 2020AnswerHello, really electrical current is an important parameter to disrupt biofilm, if you use electrophoresis DC voltage 100V with about 250mA current it will definitely disrupt biofilm but also remember about superoxide radical (toxic oxygen), which can kill your microorganisms. Unter DC voltage you will have toxic oxygen which can be decreased by superoxide dismutase or catalase. tileshop.jpg88.09 KBView22 Recommendations Damien F Meyerasked a question related to Molecular MicrobiologyCould anyone send me a FileMaker template to create a biological collection (bacterial strains, mutants, etc)?Question6 answersJan 23, 2014I am planning to create biological databases for mutants, bacterial, strains and plasmids. I want to use FileMaker pro but I am a new user and I do not know how to create my first file. A template would be of great help to see how modifying it and adding entries. Thanks to everyone.Relevant answerCristina Sarasa-BuisanAug 30, 2019AnswerHello guys! I am looking for the same!! Could anyone help me with that?View0 Recommendations Alex Ignatovasked a question related to Molecular MicrobiologyIs where any new conformation for the conclusion made by A. Hubber et al. (2004) about interchangeable functions of bacterial T3SS and T4SS?Question7 answersJul 23, 2019 Is where any new conformation for the conclusion made by A. Hubber?Hubber A, Vergunst AC, Sullivan JT, Hooykaas PJ, Ronson CW. Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system. Molecular microbiology. 2004 Oct;54(2):561-74. doi:10.1111/j.1365-2958.2004.04292.x Nevertheless, the comparisons between R7A and MAFF303099, including the identical mutant phenotypes of the respective T4SS and T3SS mutants on L. leucocephala, strongly suggest that the type IV and type III systems are interchangeable. Over the course of evolution, bacteria can adopt either, perhaps with functionally redundant effector proteins, to translocate proteins to eukaryotic hosts. Relevant answerMaxuel AndradeAug 6, 2019AnswerI agree with Alex Ignatov above \"In theory, it might happen after gain/loss of T3SS in bacterium with T4SS (or v.v.) - so, it should be a rare event most probable for rapidly evolving group of strains.” Also, I am wondering about the different signals used for secretion of effectors in T3S and T4S and how they evolved. View3 Recommendations Amir Hossain Fartashasked a question related to Molecular MicrobiologyWhat is the difference between 16S rRNA and 16S rDNA? Can genomic DNA be used in 16S rRNA method?Question17 answersFeb 5, 2014I want to use the 16s rRNA method. Can I use genomic DNA as a template in this method?Relevant answerSadegh Jamalkandi AzimzadehJul 22, 2019AnswerDear allGenetically, genes are named based on their products/functions. Therefore, there is no gene with the name of 16s rDNA. Also, 16s rRNA sequencing doesn t indicate if the RNA or DNA is used as template. Therefore, 16s rRNA sequencing can be performed based on DNA or RNA templates. Then, you should also check the downstream technique (e.g. genomics/transcriptomics). I think some authors use the term of 16s rDNA to make clear the ambiguity by mentioning that they have used DNA as template. Genome extraction ---- 16s rRNA gene amplification/seq. (16s rDNA)RNA extraction ----- 16s rRNA RNA amplification/seq. (i.e. for gene expression profiling)BestView48 Recommendations Keerthivasan Chandradoss Raaninasked a question related to Molecular MicrobiologyWhy is there an unknown band in RNA isolation of Klebsiella pneumoniae?Question6 answersMay 12, 2015The pic given is the RNA isolated from Klebsiella pneumoniae using trizol reagent. Can any one please tell me what is the bulky band at the bottom? If it is sheared rna please suggest me a way to overcome it. Even trying with all precaution about rnase I am getting that band. UVP03779 copy.jpg51.83 KBRelevant answerNourtan AbdeltawabJun 30, 2019AnswerSee attached image.jpeg96.19 KBView6 Recommendations Seung Leeasked a question related to Molecular MicrobiologyIs the cell surface of E.coli mostly hydrophilic or hydrophobic?Question6 answersDec 7, 2015Hi all,I want to attach live e.coli (MG1655) on microbeads by charge. I found two options:Aliphatic Amine Latex Beads (Thermo, A37370)Amidine Latex Beads (Thermo, A37328)Both are positively charged and in the size I want. But one is hydrophilic and other is hydrophobic. Does hydrophobicity matter on e.coli attachment? My guess was that cell surface of the e.coli is hydrophilic but I found few papers attaching e.coli on hydrophobic surfaces so I started to wander. Also, if I decided later to put lipopolysaccharides on the surface on beads, which of two beads would be easier? What would be the protocol?Thank you for your time!Relevant answerFirouzeh MorshedzadehJun 8, 2019AnswerNegative charge and hydrophobicView18 Recommendations Razibuddin Ahmed Hazarikaasked a question related to Molecular MicrobiologyTo what extent is it possible to detect M. bovis in milk samples by PCR?Question4 answersAug 18, 2016We are taking up a work on milk from peri-urban areas of Guwahati city. Although there are some literature on detection of M. bovis in milk samples, I am interested on application of molecular based techniques as well as other rapid methods. How far is it possible to detect M. bovis in milk by PCR? Other than PCR, is there any sensitive and specific rapid test for detection of M. bovis in milk samples?I shall be very happy to have the answers for food safety. Relevant answerBhairav PrasadApr 17, 2019AnswerWe are taking up a work on milk from peri-urban areas of Guwahati city. Although there are some literature on detection of M. bovisin milk samples, I am interested on application of molecular based techniques as well as other rapid methods. How far is it possible to detect M. bovis in milk by PCR? Other than PCR, is there any sensitive and specific rapid test for detection of M. bovisin milk samples?I shall be very happy to have the answers for food safety. View0 Recommendations Sayed Abdullahasked a question related to Molecular MicrobiologyPlaning PhD in Molecular Microbiology any suggestions?Question6 answersApr 8, 2019Hello everyone,I have almost completed my MS thesis degree. i worked on sRNA expression and silencing of virulent gene of avian Pathogenic E coli, hoping to publish my work soon. I want to pursue my education in the field of antibacterial therapy, virulence gene expression and antibiotic resistance. I am working on research proposal and there are many ideas like antibiotic resistance gene variation with respect to drugs and virulence gene adoption analysis . i need some suggestion about ideas and latest project ongoing related to it. it will also be good to know if some one suggest quality lab for antibiotic resistance work. ThanksRelevant answerSayed AbdullahApr 16, 2019AnswerSanchari Kundu Thank you Mam. i will look into it.,View0 Recommendations Adam Ostrowskiasked a question related to Molecular MicrobiologyPCR identification of Enterobacter sp?Question6 answersSep 13, 2016Hi, I m looking for primers specific to Enterobacter cloacae complex or the entire Enterobacter species. The idea is to distinguish its members from other enterics by PCR. Does anyone know a sequence of such pair? I found multiple ready-to-use qPCR kits, but it s too advanced for what I need and there obviously is no info on the primers used. Can anyone help please? Thanks!Relevant answerShengyu FengApr 2, 2019AnswerI also need to quantify this bacteria, and the blow primer I found in this article https://patents.google.com/patent/CN102952881B/en Hope it can help.forward primer F: 5 -CATGACACCGGTGTTTCCCCAGT-3 reverse primer R: 5 -CGGTCGGTGAAGCCCAGAACCACTA-3 . View0 Recommendations Valerie Diane Valerianoasked a question related to Molecular MicrobiologyIs there a way to prepare commercial mucin on microscope slides?Question3 answersDec 8, 2016Hello everyone,I am hoping to observe adhesion microscopically using staining. I have been doing adhesion experiments on 96-well polystyrene plates but I want to be able to visually observe the attachment.Has anybody successfully done this? What type of microscopic slide should I be looking for?Thank you for your help.Relevant answerValerie Diane ValerianoJan 8, 2019AnswerHi Ashwin Ramesh , I have had communication with another researcher regarding the procedure, with which he checked with BCA.For both of us, what worked was to bind commercial mucin to a NUNC maxisorp plate using acetate buffer (pH 5.0). A drying step was utilized after incubation with the mucin. Take note that different approaches for mucin binding to the wells are different as the composition and concentration of mucin complexes used are different. But maybe the above steps may also be helpful for you, especially with the drying step. Here is the link to our paper that details the approach we have used: Article Carbohydrate-binding specificities of potential probiotic La... Best,ValerieView6 Recommendations Muhammad Saleem Kalhoroasked a question related to Molecular MicrobiologyWhy lactobacilli have intrinsic resistance toward Vancomycin?Question4 answersSep 25, 2016Why lactobacilli have intrinsic resistance toward Vancomycin?Relevant answerAlaa Hani Al-CharrakhDec 14, 2018AnswerIt s well-known that in several species of LAB, the terminal d-alanine residue is replaced by d-lactate or d-serine in the muramylpentapeptide, preventing vancomycin binding and therefore becoming resistant to this antibiotic.RegardsView36 Recommendations Joanna Drabińskaasked a question related to Molecular MicrobiologyHow to prepare a solution of 2-ketogluconic acid from 2-ketogluconic acid hemicalcium salt hydrate?Question2 answersDec 4, 2018 I am trying to prepare 2-ketogluconic acid from 2-ketogluconic acid hemicalcium salt hydrate (Sigma). I found a protocol in: SWANSON, Britta L., et al. Characterization of the 2‐ketogluconate utilization operon in Pseudomonas aeruginosa PAO1. Molecular microbiology, 2000, 37.3: 561-573. : A calcium-free solution of 100 mM 2-ketogluconate was prepared as follows: 1.45 g of 2-ketogluconic acid salt was dissolved in 48 ml of distilled water, heated to 70 °C for 10 min and 12 ml of 10.5 M potassium phosphate buffer (pH 7.0) was added. It seems to me it is implausible to prepare a 10,5 M potassium phosphate buffer, because it would require dissolving more KH2PO4 and K2HPO4 then it is plausible. What would be the correct concentration of the potassium phosphate buffer to obtain the desired solution of 100 mM 2-ketogluconate? Are there other easy ways to obtain such a solution? Relevant answerJakub GrzesiakDec 5, 2018AnswerI would proceed according to the protocol. The phosphate buffer is given in excess to make sure that all the calcium is precipitated. The reaction would not be at 100% efficiency if it was conducted at stoichiometric ratio. Solubility of potassium phosphates is relatively high (150g/100ml for K2HPO4 at 20C). I suggest heating the buffer solution to dissolve it completely.View3 Recommendations Abdul Rahimasked a question related to Molecular MicrobiologyHas anybody seen coagulase negative staphylococcus aureus before?Question12 answersMar 25, 2015And good day. I just want to ask. Is it anyone of you facing coagulase negative Staphylococcus aureus before? Actually I test my Staphylococcus aureus isolate for clot formation (coagulase test) by slide test and tube test (4 hour incubation at 37°C degree) and still it not shown any clot formation. Is it any possible s? Aureus exist as coagulase negative strain? Thank you.Relevant answerAala’ A. ChmaghNov 30, 2018AnswerMr UelintonYou need to use other primer for coa gene amplification, because of there are many primers couldn‘t amplify all coa alleles due to it high polymorphism geneView4 Recommendations Zeynab Farajiasked a question related to Molecular MicrobiologyWhat is the best protocol to do RAPD analysis for studying genetic diversity of UPEC?Question6 answersAug 2, 2016I want to study genetic diversity of Uropathogenic E. coli  .I  DNA extraction methods for E.coli by boiling.but i don t band in elektroforez.Relevant answerRūta PrakapaitėNov 10, 2018AnswerDear Zeynab Faraji and everyone with the particular interest,The RAPD is a very demanding technique, as it requires a lot of precision and the right PCR conditions to be maintained. Firstly, if the RAPD is failing, I would suggest to perform the gradient PCR. I had assessed RAPD with DNA extracted by boiling method and the results were really great, of course, after performing the gradient reaction and finding the best conditions. In addition, another cheap, repeatable and more reliable method for the genetic diversity of UPEC is ERIC-PCR. Good luck.RūtaView6 Recommendations Ayyapruk Moungprayoonasked a question related to Molecular MicrobiologyWhat is the theoretical yield of lactic acid fermentation from sucrose?Question8 answersJan 12, 2017I used sucrose as a substrate for lactic acid fermentation by Bacillus sp.. But, I could not find the data that showed me about theoretical yield of from sucrose. Thank you very much.Relevant answerChristopher IbenegbuSep 30, 2018AnswerTheoretical yield of lactic acid for homofermentative organisms is 100% (1g lactic acid per g glucose). That is 2 moles lactic acid per mole of glucose. For xylose, it is 1.67 moles of lactic acid per mole of xylose.BUT, in the presence of carbonates used in neutralisation during fermentation, these yields can be up to 120% ( 100%), due to the utilization of the carbonates as extra substrate.View6 Recommendations Priyanka Mishraasked a question related to Molecular MicrobiologyMicrobial Lag phaseQuestion28 answersAug 24, 2018Microbial lag phase is very important stage of bacterial growth to understand the physiological and regulatory process responsible for adapting to new environment. But very less is know about this phase of microbial growth. As per textbooks, lag phase allows the adaptation required for bacterial cells to new environmental conditions. This stage include the repair of bio-molecular damage and the synthesis of cellular components necessary for growth. This shows cells are metabolically active during lag phase, but are they just repairing and synthesizing bio molecules? Relevant answerAbhijeet SinghAug 30, 2018AnswerChronologically-1) Alireza Mordadi - No quorum sensing is not a neither a signaling mechanism nor it regulates the deterministic factor for cells size determination or trigger for cell division. It is the mechanism which overall related with the population density in a particular environment. Basically it is radar homolog in bacteria. It might indirectly involve in the aforementioned, but not directly. Therefore, recommending quorum sensing (a wider topic) for a specific question is not supported.2) Jay Sperry - since the discussion was not related to the biofilm but a bacterial growth in normal growth protocols, so I tried to relate the your previous comment to the context. And again it does not fit well, as you mentioned only one bacteria without mentioning any biofilm formation. E. Faecalis forms biofilm after 14-15 days of incubation (PMID: 27095620). So, what happens meanwhile, do cells divide or they remain in lag phase? And if we talk about E. faecalis biofilms, SEM showed E. faecalis growth at all times (PMID: 27095620).Nevertheless, biofilms are special microbial structures, where the conventional growth curve does not applies. Therefore, involving biofilms in this discussion is like comparing apples and pears. * I agree with your comment that bacteria might have different physiological growth phases, and it has been considered previously in the discussion above. Each species has its own growth rate and ofcourse cells requires time, a certain cell size and mass to initiate the division. But should we call it lag phase? View33 Recommendations Yuan Liasked a question related to Molecular MicrobiologyHow to design the primer for gene deletion using RED system?Question1 answerAug 19, 2018 Recently, I have been used the RED system to delete the target gene in EPEC. According to the paper, the primer uesd to amplify the selectable marker (cat) is flanked by 50 bp of tir sequence.(Campellone K G, Giese N, Tipper O J, et al. A tyrosine‐phosphorylated 12‐amino‐acid sequence of enteropathogenic Escherichia coli Tir binds the host adaptor protein Nck and is required for Nck localization to actin pedestals[J]. Molecular microbiology, 2002, 43(5): 1227-1241.) Specifically, the primer provided in this paper were: 1. P-tir5 KO ATGCCTATTGGTAACCTTGGTAATAATGTAAATGGCAATCATTTAATTCCGCGGCCGCATGAGACGTTGAT 2. P-tir3 KO TTAAACGAAACGTACTGGTCCCGGCGTTGGTGCGGCATTTACAGAACTTAGCGGCCGCTTTCGAATTTCTGC   Since I have no experience in gene deletion in EPEC, I have some confusion about the design of primers. I thought the sequence of P-tir5 KO should begin with the initiation codon of tir and cat, which were splited by Not1 site ; the reverse sequence of P-tir3 KO should be from the stop codon of tir and cat. However, there are no results when I use these sequences to blast. Therefore, I am very confused. Would someone please give me some help about the design of primer used to delete the targeted genes?  Thanks very much. Relevant answerWieslaw SwietnickiAug 20, 2018AnswerHi Yaun, Not necessarily. You design primers to amplify fragments constituting missing part of the gene. The hooks of 50 bp each are for anchoring it to the rest of the DNA. In the simplest case, hook left- fragment left- fragment right-hook rightLength of the hook is determined by the recombination system. Without RED, you need 1 kb on each side of the gene (hook length) plus marker (kan/amp) to select for the right clone. With RED, you need 50 bp on each side of the gene and marker.For in vitro systems, you will need linear DNA designed as before with the selection marker and typically a exonuclease inhibitor. You can do it by electroporation without RED using 1 kb hooks.best luck,WesView0 Recommendations Luisa Fernanda Posadaasked a question related to Molecular Microbiology¿What´s the best method for microbial DNA extraction of banana roots for metagenomics processes?Question9 answersAug 9, 2016I want to know What´s the best method for microbial DNA extraction of banana roots because i need microbial root DNA of banana for metagenomic studies. Relevant answerMatt GacuraJul 18, 2018AnswerI have utilized CTAB with great success in extracting fungal DNA from leaf litter at a very low cost. The MOBIO powersoil kit has also worked very well with leaf litter.View10 Recommendations Mushtak T. S. Al-Ouqailiasked a question related to Molecular MicrobiologyDear all, does some one work in the use of real time PCR in the detection of metallo-beta-lactamases in klebsiella spp?Question6 answersJun 8, 2018The requirement for updated and modern researches regarding the use of real time PCR in the detection of metallo-beta-lactamases in klebsiella pneumonia and pseudomonas aeruginosa...Relevant answerAnthony Ayodeji AdegokeJun 13, 2018AnswerAre you doing direct detection from samples? If not, it s as simple as ordinary detection using primer based detection, rt master mix, no probe, non template control, and your positive control or standards, etc. You can also quantify using your standards to comparatively determine the quantity of the genes in your samples. This would be done automatically by your thermocycler, but you have to determine and insert the copy number calculated using the avocadro constant for your standards. Qualifications are only reasonable for sample based detection and less irrelevant for isolate based. View22 Recommendations Jason Saredyasked a question related to Molecular MicrobiologyCan you do PCR on a T4 ligated plasmid?Question5 answersSep 5, 2016I am working on a virus that we have in a bluescript vector. The end goal is to rtPCR patient data but to test the primers I d like to use our viral genome without blue as some of the primers will span the blue vector as well making the product 3kb longer. So I tried to cut it, run on a gel, extract the viral band, then ligate that. Then I run that against the cut viral band and an uncut plasmid that is approximately the same size as the virus, assuming the major band on the uncut plasmid to match the size of a single viral genome religated and supercoiled. Then extract just that band. Doing PCR though anything that spans the cut site isn t amplifying. The virus was put into the vector with the same cut site on both sides (BamH1)Either way I can use the original in bluescript, but I d like to have matching sizes between the test PCR and the background transcript in the patient data as a supplemental picture. My question is after doing T4 ligase, will denaturing the DNA break the strands at the BH cut site? Is there an easy way to ensure it doesn t break apart? We can t transform cells since we don t have a selection marker after cutting out bluescript.Relevant answerJason SaredyMay 25, 2018AnswerSecond update: I heard back from the lab that they figured out what may have been the problem. The BamHI HF batch we used apparently may have been the problem as he finally sat down to do some more basic troubleshooting. I would be surprised if it was a HF vs nonHF problem and not a batch problem of the HF enzyme, but they ordered a new nonHF version and it worked fine. I think that was one of the only steps we didn t try before moving to infusion cloning since it was clearly cutting. View0 Recommendations Catia Cillónizasked a question related to Molecular MicrobiologyMolecular microbiology for infecton diseases, is this the future? Question4 answersMay 4, 2018Some experiences to share with new molecular tecniques on diagnosis infectious diseasesRelevant answerOliver NolteMay 4, 2018AnswerAt this time, the classical approach in infectious disease diagnostics is still culture based for the majority of indications. For some indications, molecular assays are available and these are being increasingly used in the laboratories. Many companies already offer multiplex panels for a syndromic diagnostic testing approach. At this year’s ECCMID conference, we have seen a lot of cartridge multiplex PCR or nucleic acid amplification formats, enabling to test for diarrhea pathogens, respiratory pathogens, etc. Saying this, my first answer is yes, the future in infectious disease diagnostics is molecular microbiology. However, nucleic acid amplification techniques, most likely even in POC settings, will serve as first line diagnostics, followed by culture were necessary. I therefore don’t expect a complete replacement of clinical microbiology by clinical molecular biology. However, certainly the clinical microbiology lab will undergo dramatic changes in view of the shift to molecular techniques on one side and to automation systems for culture (i.e. WASPLab, Kiestra TLA) on the other side. View1 Recommendation Eddie Fadeevasked a question related to Molecular MicrobiologyLooking for on going projects of marine microbial observatories?Question4 answersSep 20, 2016In order to gather knowledge regarding state of the art tag sequencing methods used in the field of microbial oceanography, I am looking for on going projects of marine microbial observatories/recently published studies regarding time series of marine microbial communities.Thanks in advance!Relevant answerEddie FadeevApr 18, 2018Answerhttps://www.sciencedirect.com/science/article/pii/S1369527417302035?via%3DihubView0 Recommendations Pavel Parilovasked a question related to Molecular MicrobiologyEnterocytozoon Hepatopenaei spp. (EHP spores) and its stability against chemical disinfectants and UV light?Question5 answersMay 1, 2016Dear colleagues,Does anybody know something about spores of Enterocytozoon Hepatopenaei especially with regards to its stability against chemical disinfectants and UV light?I would be appreciate for any information/links/articles or thoughts about this issue.I have already found that it is quite new organism and there is not much researches dedicated to this problem.Thanks!Relevant answerJithendran K.P.Feb 28, 2018AnswerI will recommend to read the latest article detailed below:doi:10.1016/j.aquaculture.2018.02.039Bioassay for spore polar tube extrusion of shrimp Enterocytozoonhepatopenaei (EHP)View6 Recommendations Mustafa Vohraasked a question related to Molecular MicrobiologyNeed a topic to study on RNA from human blood ?Question3 answersDec 21, 2017Need a topic to study on RNA from human blood where i can utilise QIAamp RNA Blood MINI KIT?This may sound a bit strange but I am looking for a good study topic where i can utilize Qiagen Qiaamp RNA Blood Mini Kit for some good use.Hello EveryoneThe thing is i am working in a district government hospital as a Microbiologist. Here along with routine sample analysis we are also developing a molecular microbiology lab where we are developing protocol for identification of human pathogens and detecting drug resistance. We have a QIAGEN QIAcube, Rotor Gene Q, QIAxpert, Serum HPLC and PCR at our disposal.Now during our initial setup we had asked QIAGEN to provide all the necessary kit which would be required by our lab. That s where they provided us with 4 Boxes, 50 reaction each of Qiagen Qiaamp RNA Blood Mini Kit. We are not sure where to put good use of it. We are still not studying human blood RNA. We do study viral RNA but for that we use QIAamp Viral RNA mini kit. Now since we have this RNA Blood kit, we are planning to use it for some useful purpose which would help further development of our laboratory. We have an attached pathology lab where we can get blood sample for any disorder. So i need help from you on suggesting me based on your experiencea) a topic which i can look upon or b) a target RNA in human blood for disorder/Cancer orc) Any Viral RNA which can be recovered by Qiagen Qiaamp RNA Blood Mini Kit from blood ord) Any other suggestionby which i can utilize RNA Blood Mini Kit Limitation: I am looking for a single or may be just 2 RNA target to study. We have Reverse transcriptase kit and Q-PCR at our disposal. We can also purchase primer for a any target CDNA. Designing a single probe for Q-PCR is possible but i don t have budget for multiprobe study. If the target gene could be studied by SYBR green kit than that would be great.Thank youLimitation: I am looking for a single or may be just 2 RNA target to study. We have Reverse transcriptase kit and Q-PCR at our disposal. We can also purchase primer for a any target CDNA. Design probe for Q-PCR is possible but i don t have budget for multiprobe study. If the target gene could be studied by SYBR green kit than that would be great.Relevant answerPrabhash JhaDec 22, 2017AnswerHi Mustafa,First, you need to select the disease based on the sample availability. And then the second step will be to identify the gene targets for RNA experiments, this can be done in silico using the publicly available gene expression datasets (however this requires bioinformatic skills). Other way to narrow down to the targets may be to find out some good publication (literature survey) which have identified novel target genes for the particular disease but the experimental validation is yet to be done on human samples and use these target genes for your RNA experiments after acknowledging that particular article.Hope this helps.View5 Recommendations Lorenzo Mazzoliasked a question related to Molecular MicrobiologyHow you stimulate RAW 264.7 macrophage?Question9 answersAug 31, 2016I am using a protocol with 16 hours of stimulation with LPS (100ng/ml) and then 24 hours of treatment with my samples. I remember that I found an article where did such a thing, but I can not find it. I need it for a quote, but in all the articles I ve read recently is made a cotreatment with LPS and samples to be tested.Relevant answerKaja ŻuwałaNov 27, 2017AnswerDear Milena1. How many cells must be plated to obtain an optimal stimulation?Our lab found 20 000 cells per well in 96-well plate as most optimal but that it doesn t mean it is the best option for your experiment2. What parameter do you use for normalize Nitrite results?I am not sure I understand, but we simply perform Griess assay with a standard curve and then normalise to the sample treated with LPS only.3. With is the solvent for LPS and Do you sonicate LPS?We buy one from Sigma and dissolve it in PBS, store small aliquots in the freezer and never reuse aliquot which has already been thawed up.4. Do you see some morphological changes in activated raw cells?Only if the LPS concentration was so high that it was toxic. Otherwise not. 5. During LPS treatment do you use FBS?Yes6. Do the cell passages influence LPS stimulation?Yes, the best cells are of low passage number and after passage 10-15 we observe loss of NO productionGood luckKajaView6 Recommendations Iffah Inani Hashimasked a question related to Molecular MicrobiologyHow to sterilize properly using autoclave (for molecular / microbiology works)?Question5 answersOct 9, 2017Hi, is it right how I sterilize these items:1. Pipette tips box: Close the box and autoclave or should I open it slightly to allow steam to penetrate.?2. PCR tubes/microcentrifuge tubes: Place it in glass beaker and cover with aluminium foil.3. Mortar and pestle: Wrap with aluminium foil and oven dry for 2-3 hours at 200 degree celsius? Can I autoclave?4. Stainless steel (forceps, scissors etc): wrap in aluminium foil, then autoclave or oven dry at 180 degree Celsius? I read somewhere on internet that for items like PCR tubes that are DNase or RNase free, it is not necessary to autoclave, but with our lab condition, I am not confident with it sterility, and I opt to autoclave it again. Relevant answerSharifah Farhana Syed-Ab-RahmanOct 9, 2017AnswerHi Iffah,Here are the answers for your questions, based on my experience working in the lab:1. Close the box properly, make sure no water can go inside the box then autoclave it. You can also use tape to make sure it is sealed properly.2. Yes, that s what we usually do. 3. Wrap them with aluminium foil, autoclave it, take them out and let it sit in the oven until it is dried.4. Yes. Although the microcentrifuge and PCR tubes are DNase and RNAse free, they are not sterilise. We usually autoclave all these items especially if you are working with biological stuff that can easily contaminated.Hope it helps.View20 Recommendations Alireza Mordadiasked a question related to Molecular MicrobiologyExternal death factorQuestion2 answersJul 19, 2017in my knowledge, there are three identified external death factor for bacteria, is it true?those have a limited range action, is there a broad range of them?Relevant answerAlireza MordadiJul 20, 2017AnswerI discuss about other things. EDF is a quorum sensing signals that starting programmed cell death in bacteria and it isn t extra cell toxin. EDF in some way caused unstable toxin-antitoxin complex in bacterial cell.   View0 Recommendations Hans Chin Aroraasked a question related to Molecular MicrobiologyDNA extraction with phenol-chloroform - DNA stuck in phenol layer?Question5 answersJan 24, 2017I recently performed a DNA extraction for a microbiome project from cotton rectal swabs and urine pellets using phenol-chloroform. Briefly:1. Swabs were incubated with a mutanolysin/lysozyme enzyme cocktail for 1h @ 37C2. Added glass beads, phenol:chloroform:isoamyl alcohol (25:24:1) and 20% SDS, then agitated (TissueLyzer/bead beater) and centrifuged @ 14k rpm x5 min. 3. Aqueous layer transferred to a clean tube, and added 3M sodium acetate and ice-cold isopropranol at -20C overnight.The next day, no pellet! I pulled off the supernatant carefully (assuming there might be a pellet I couldn t see) and re-constituted in water, ran a PCR and a 1% agarose gel but no DNA bands! I ran the sample on the UV-Vis (Nanodrop) and got nothing (no peaks, no DNA). I also tried UV-Vis on the supernatant and got what looks like a phenol peak at 270 and also a second peak at 260, which I think is DNA (see attached). My interpretation is that the aqueous supernatant (with NAOAc and isopropanol) still has both phenol and DNA in it, the former being a technical error I suspect. I ve tried adding more NAOAc or isopropanol or even repeating the phenol-chlor extraction with aliquots of this supernatant, but to no avail. Does anyone have suggestions on how to pull the DNA out of this solution?Nanodrop data.pdf157.01 KBRelevant answerAlexander Sasha VlassovMay 24, 2017Answerorganic extraction can be tricky. try PureLink microbiome kit- it has protocols for swabs and other View0 Recommendations Philip Lyonsasked a question related to Molecular MicrobiologyHow many cycles in 16S illumina sequencing once adapters added?Question2 answersJan 10, 2014I am performing a 16S microbiome study, and have sent my samples to a lab for NGS using illumina MiSeq, until our own MiSeq here is up and running. I have received pictures of the preliminary microbiome PCR gel (25 cycles) and there is a strong band of the expected length (approx. 300bp) in all of the samples analyzed. A second PCR was then performed with sequencing adapters added, which displayed very faint bands on the gel, at a slightly higher molecular weight (due to the adapter integration).I asked the sequencing company whether this was due to reduced number of cycles at the library prep stage and they confirmed that this was the case, with only 5 cycles to avoid PCR bias. Is this enough, or are more cycles than 5 usually used? The genomic DNA used for PCR template 16S amplification was extracted from the fish gut, and measured between 50-300ng/ul with Qubit analysis.Relevant answerDavid HuybenMar 14, 2017AnswerThis is a late answer, but I have had success using 10 cycles. I have seen as low as 8 cycles. I have heard there are new Illumina primer barcodes that can be used in 1 step instead of 2 step PCR, which would save a lot of time and money. However, I haven t found them yet.View0 Recommendations Rakesh Maharjanasked a question related to Molecular MicrobiologyHow do I transform bacteria?Question6 answersMar 10, 2017I cloned my gene of interest in pET-22b vector. I could transform DH5α E. coli strain. But I am not being able to transform expression strain of E. coli. My transformation protocol is; thaw the competent cells for 30 mins in ice after adding vector, heat shock for 45 sec at 420C, place back in ice for 2 mins, add 500 µL SOC, incubate at 370C and then transfer to LB plate. And I am using ampicillin at concentration of 150 µg/ml. So, why do I get bacterial colonies for DH5α but not for other strains?Relevant answerBhoj R SinghMar 14, 2017AnswerIt may be helpful to read the book at the link.Book Microbial Genetics Questioned to UnderstandView0 Recommendations Samira Fattahasked a question related to Molecular MicrobiologyWhat are the disadvantages of isolating bacterial genome by direct pcr kit?Question4 answersDec 19, 2016I need to know the purification quality of these kitsRelevant answerChristopher SloweyFeb 28, 2017AnswerIf you are worried about the purity of your sample, there are commercial products available to remove contaminating human DNA and significantly amplify signal:  https://www.researchgate.net/publication/298064100_Broad-Range_Microbial_DNA_Isolation_from_Clinical_Specimens_for_Universal_PCR_DiagnosisConference Paper Broad-Range Microbial DNA Isolation from Clinical Specimens ...View0 Recommendations Edward Jonesasked a question related to Molecular MicrobiologyIs there an enzyme that can be used to break down GFP or RFP?Question1 answerFeb 18, 2017Designing a circuit in E. coli that expresses GFP when carbon monoxide is present via a modified CooA protein and modified lac operon. RFP is then expressed when there is no carbon monoxide. I need to break down the other when one is expressed so that the signals do not interfere with each other but struggling to find a protein that will break it down.Relevant answerAlexandra JohnsonFeb 20, 2017AnswerThere are GFP variants that have been made unstable by adding a short peptide tag that targets them for degradation so they don t hang around in the cell for very long. I ve never worked with them personally, and maybe someone s improved on it since this paper, but it s probably a good place to start. It also seems like the easiest option for modifying existing constructs. Article New Unstable Variants of Green Fluorescent Protein for Studi...View5 Recommendations Innocent Onunkwoasked a question related to Molecular MicrobiologyI m working biosynthesis of PCA antibiotics, but I want to do an Isolation of the primary organism, P.aeruginosa, how do I go about it?Question5 answersDec 19, 2016I m isolating P. aeruginosa from an oil spilled soil using Mineral salt medium, thereafter go on to isolate the PCA from the organism but am not getting positive result, which other way should I go about that? I also want to know know the chemosynthetic way to produce this compound, PCA antibiotics. ThanksRelevant answerInnocent OnunkwoFeb 18, 2017AnswerThanks a lot, Marielsa Gil. I found your recommendation and directions very helpful. I Will get back to you on the progress soon. Thanks once more.View0 Recommendations Aysha Haiderasked a question related to Molecular MicrobiologyWhy is DNase I added to the bacterial cell suspension?Question4 answersFeb 9, 2017If I want to lyse bacterial cells and extract bacteriocin, should I add DNase I to the suspension? will it have any effect on the concentration of bacteriocin or DNase I will only cleave DNA?Relevant answerMichael J. BenedikFeb 9, 2017AnswerThe role of the DNase is to reduce viscosity, otherwise your lysate is highly viscous and things get trapped. Sanitation or some other treatment that disrupts the DNA can be used instead of DNase, or if you are doing a gentle detergent lysis then the addition of DNase just makes the subsequent steps easier. It should not have any effect on the bacteriocin concentration but might make it easier to purify. By the way, many bacteriocins are extracellular and you can purify them without lysing the bacterial cells. You might want to determine whether that is the case with yours. View6 Recommendations Samuel Aziegbemhinasked a question related to Molecular MicrobiologyDoes anyone have a protocol for antibiotic susceptibility testing for non culturable fungi ?Question6 answersAug 8, 2016I would like to do susceptibility tests on non culturable fungi isolates from clinical and environmental  samples.Relevant answerSamuel AziegbemhinJan 30, 2017AnswerThank you Umar.View0 Recommendations Laura Masonasked a question related to Molecular MicrobiologyIs there an ideal number of samples for MiSeq?Question13 answersJan 8, 2017I plan to use the Illumina MiSeq platform to sequence cDNA of a transcriptome from soil bacteria and fungi. Relevant answerLaura MasonJan 26, 2017AnswerThanks for your help - I definitely need to do more researchView0 Recommendations Ian Cohnasked a question related to Molecular MicrobiologyHas anyone used lysozyme prior to bacterial PCR? What formulation/from what company? Have you done so without any downstream purification?Question5 answersJan 18, 2017I would like to perform PCR on crude bacterial lysate. Has anyone successfully done so using lysozyme, and if so what formulation was used?Relevant answerMaría Elisa PavanJan 25, 2017AnswerUsing a fresh culture, you could try resuspending a few colonies in 150 µl TE buffer (10 mM Tris-HCl, 1 mM EDTA), boiling at 98ºC during 10 min in the thermocycler and using the supernatant as DNA template (5 µl) for amplification.  It works well with Bacillus anthracis.View0 Recommendations Joan Peñaasked a question related to Molecular MicrobiologyI am looking for a protocol for DNA extraccion of Staphylococcus, without using of lysostaphine enzime?Question5 answersJan 25, 2017I know the lysostaphine is the easy way to extract DNA from Staph, but it is expensive. I prooved already the simple boiling way but it does t work.Relevant answerMaría Elisa PavanJan 25, 2017AnswerUsing a fresh culture, you could try resuspending a few colonies in 150 µl TE buffer (10 mM Tris-HCl, 1 mM EDTA), boiling at 98ºC during 10 min in the thermocycler and using the supernatant as DNA template (5 µl) for amplification.  It works well with Bacillus anthracis.View5 Recommendations Sabrina Diemertasked a question related to Molecular MicrobiologyUsing resazurin solution as an anaerobic indicator?Question16 answersJan 23, 2017I m doing some anaerobic microbial experiments, in which I m using an anaerobic hood for preparation of cultures and plating for enumeration. I put my plates into sealed jars, and remove them from the anaerobic hood to incubate. I think that the jars are maintaining anaerobic conditions, but I want to use an indicator as proof. I know I can buy indicator strips, but I have resazurin in my lab, so I made up a solution (30 mg/L in deionized water), and left it in my anaerobic hood to deaerate. However, after over 24 hours in the hood, the solution was still blue. As I understand it, the solution should turn pink initially (and irreversibly), then clear when under anaerobic conditions. According to my oxygen/hydrogen probe in the hood, conditions were always under 1ppm oxygen.Am I missing something here? Do I need to provide alternative reducing conditions (somehow?) in the solution initially so that it turns pink? Or would purging the solution with nitrogen ensure that my solution is fully anaerobic? Any thoughts would be greatly appreciated!Relevant answerValter TandoiJan 25, 2017AnswerI have been working long time with Resazurin:  it clearly show anaerobic conditions  only when there is total absence of oxygen. Also the pH is important for Resaruin to become clolorless. The point is that flushing with nitrogen does not mean having total absence of oxygen. When I worked with liquid media, the addition of sodium sulphide caused the riduction of all the oxygen and the medium became colorless. Working with anaerobic jars, you have to add some small bags: the compound inside the bag reacting with added water, produces hydrogen, that by a catalizer bag  located in the jar, allowes the total reduction of oygen. In these conditions, the small strips of resazurin indicator become colorless. The nitrogen alone, even at high purity, contains always minor amounts of oxygen: the resazurin never will turn colorless if you do not add a compound able to transform the residual oxygen in H20. So if you need strict anaerobic conditions, you must add a chemical that destroys all the residual oxygen present.See these papers:Tandoi V., Di Stefano T., Bowser P.,Gosset J.M. And Zinder S.H. (1994): Reductive dehalogenation of chlorinated Ethenes and halogenated ethanes by a high rate anaerobic enrichment culture. Environmental Science and Technology, 28, 973-979.Gatell X.M., Tandoi V., Gossett J.M. and Zinder S. H. (1995): Characterization of an H2 utilizing enrichment culture that reductively dechlorinates tetrachloroethene to vinyl chloride and ethene in the absence of methanogenesis and acetogenesis. Applied and Environmental Microbiology, 61, 3928-3933.View40 Recommendations Nguyen Quiasked a question related to Molecular MicrobiologyHow to compare the number of bacteria in different types of samples based on qPCR results?Question10 answersJan 19, 2017I have three types of samples including liquid samples (water, milk), solid samples (feed, feces) and aerosol samples. After quantifying by qPCR, I have the copies number of each sample. However, I don t know how to compare the number of bacteria among these samples because of the differences in their characteristic and difference in unit (g vs ml) as well. Could anyone give me suggestion? Many thanks.Relevant answerJai GhoshJan 20, 2017AnswerLet me know why do you want to compare the number of bacteria. As per the information given by you, it is a redundant exercise. View10 Recommendations Rishabh Kaushikasked a question related to Molecular MicrobiologyWhat should be the minimum sample size for comparison of rhizospheric bacterial communities using illumina platform?Question6 answersJan 17, 201716s paired -end sequencing using illumina platform.Relevant answerJosé Francisco Cobo DíazJan 19, 2017AnswerAt least 3 soils samples (or replicates) per plant, but it is better if you have more.View5 Recommendations Muhammad Saleem Kalhoroasked a question related to Molecular MicrobiologyIdentify lactic acid bacteria (lactobacillus) up to species level without molecular techniques?Question8 answersJan 19, 2017I have isolated lactic acid bacteria isolates and through basic characterization I found that they belong to lactobacillus genus is there any biochemical method which can differentiate species of isolates before I do 16S rDNA analysis Relevant answerHarsh PanwarJan 19, 2017AnswerDear Saleem, I suggest you to directly proceed for species specific PCR or 16S rRNA based sequencing. Using biochemical methods may not be useful and conclusive.View10 Recommendations Ladan Khodaparastasked a question related to Molecular MicrobiologyHow to screen a large collection of different bacterial isolates with lowest contamination risk (liquid LB medium)?Question7 answersJan 17, 2017How to screen a large collection of different bacterial isolates with lowest contamination risk (liquid LB medium)? and,  Do you have any suggested how to keep them in -80 with limited space?We will have a large collection of different bacteria together which we need to screen them. Although, I know that one of the possibility is keeping them in 96 well plates to not get too much space but anyone has any other suggestion? Even if we sealed them by stickers, we will have the risk of contamination. Do you have any other suggestion in this regards?ThanksRelevant answerWerner SolbachJan 19, 2017Answerthe best is to use small glass capillaries as container for frozen isolates (20% glycerol or DMSO).View7 Recommendations Anwar H. Sharafaddinasked a question related to Molecular MicrobiologyWhat is the better way to Evaluate extracellular enzyme in Congo Red Plate?Question7 answersJan 12, 2017I m studying the ability of extracellualr enzymes production for some fungal strains using congored plates. What is the best way to calculate the enzyme index. what I m trying to use now eitherEI=diameter of hydrolysis halo zone/diameter of colony OR area of hydrolysis halo zone/area of colonyRelevant answerPaola QuatriniJan 19, 2017AnswerIt depends how you obtain fungal colonies. Do you start from one spore or just a loopful of mycelium? in this second case the colony diameter or area is not indicative of growth. Thus I would suggest to measure only the decoloration halo diameter excluding the diameter of the colonyView7 Recommendations Viralkumar Panchalasked a question related to Molecular MicrobiologyWhat is the big peak at 5S position of Staphylococcus aureus RNA profile? Is this normal observation?Question6 answersJan 17, 2017RNA profile for all my samples shows strange big peak at 5S position. Should I use these RNA samples for downstream experiments? Does anyone know if this is common with Staphylococcus aureus. Total RNA was isolated by using RNA protect (Qiagen) for stabilisation of RNA. RNeasy kit was used for extraction. I have also noticed that the concentration of RNA is differs with the instruments used. Nanodrop gives higher concentration than Bioanalyser for the same samples. Please let me know what you think regarding the peak shown in attached image. I have very limited experience with RNA and seeking for experts help. I am happy to provide any other detail you might want to know. Thank you very much. RNA profile.PNG47.65 KBRelevant answerTanino Giuseppe AlbaneseJan 19, 2017AnswerFrom the position of the small peak close to the 5S, I would say that you are detecting the tRNAs, so nothing to worry about. You can run the sample on a PAA gel next to an appropriate ladder for confirmation, the bands should migrate between 70-90bp. I would rather be concerned about the great imbalance of extracted 5S and 16/23S. Is the kit/protocol you used designed for specific isolation of small RNA?View12 Recommendations Laura Marise de Freitasasked a question related to Molecular MicrobiologyDoes anyone know a protocol to quantify hydroxyl radicals and singlet oxygen formation in bacteria that DOES NOT involve microscopy?Question4 answersJan 11, 2017I have been looking for a protocol to quantify ROS formation in bacteria using a spectrofluorimeter, NOT a fluorescence microscope, but I failed to find one.I have APF and SOSG (singlet oxygen sensor green) as probes.Relevant answerGregory BarshteinJan 18, 2017AnswerI hope that following publication will be helpfulhttps://www.researchgate.net/publication/5544216_Comparison_of_fluorescence-based_techniques_for_the_quantification_of_particle-induced_hydroxyl_radicalsArticle Comparison of fluorescence-based techniques for the quantifi...View5 Recommendations Fahimeh Mohammadiasked a question related to Molecular MicrobiologyIs it necessary to do the standard curve before we conduct the relative quantitative real-time PCR for microorganism?Question6 answersJan 17, 2017hiWhat is the prerequisite before me conduct relative quantative real-time PCR?Relevant answerLaurence Stuart Dawkins-HallJan 17, 2017AnswerDear FahimehIn short yes: The principal  reason for this is that a standard curve is designed to let you know how efficiently your primers work. This is required for calculation of relative expressionThe most popular method for calculating relative expression is the so called Delta Delta Ct or livak method: That assumes a perfect doubling during the exponential phase of amplification and for that to apply and render your relative expression value(s) valid the efficiency of your primer must be ideally 95% to 105%) or at the very least 80% if you are dealing with orders of magnitude expression difference rather than for example 1-2 fold in crease in expressionFind attached the original Delta Delta Ct paper by Livak explaining the rationale behind the methodAlternatively, if your primer efficiencies fall between 80% and 90% you also need to know the exact value in order to factor this in to your  relative expression calculation using something called the pfaffl methodFind attached the original paper by Pfaffl and a chapter from a book explaining his methodFinally, Primer efficiency deduced by a standard curve is effected by primer efficacy in turn rooted in optimal primer design; Secondary structure in your target affected by high GC content ( 70%) or consecutive runs of G/C residues ( 4 in any given loci); High AT content, both reducing replication efficiency; length of amplicon (~ 100bp is best for maximal amplification efficiency); copy number of your target; whether your primers are 3 biased ( RNA degrades from the 5 end and thus primers situated within 1kb of the 3 terminus of your transcript, leading to higher and more accurate representation of those regions of mRNA in your cDNA library, especially if your RNA is visibly degraded) and purity of your RNA and thus efficiency of cDNA synthesisThus, best practice would indicate that you do not just perform a standard curve for each new set of primers but you replicate this standard curve for each new biological replicate, i.e. cDNA sample, although most person (s) in reality tend to just calculate efficiency for new primer pairs; especially if your starting RNA has a 260/280 1.7 (low protein contamination including potentially RNASes)  and a 260/230 ratio 1.0 (implying low salt contamination and in particular GITC bleeding through from the lysis buffer which will inhibit reverse transcriptases); and also if RNA integrity has been verified by agarose gel or better still Agilent Biochip pfaffl method.pdf218.94 KBPractical Biostat day 1 hand out.pdf1.85 MBchapter-3-pfaffl.pdf218.94 KBDelta Delta Ct analysis_Livak_2.pdf488.61 KBView8 Recommendations Mohamed issam Draiefasked a question related to Molecular MicrobiologyWhy I find almost my lactic acid bacteria collection inhibits L. innocua while only half of my collection inhibits L. monocytogenes ?Question4 answersJan 16, 2017Lactic acid bacteria producing bacteriocinRelevant answerAlaa Kareem NiamahJan 16, 2017AnswerHiThe resistant bacteria against lactic acid Bactria different among spices of bacteria. example five strains of   L. monocytogenes  some strains inhibition and no inhibition either strains . The resistant bacteria  depends on several thingsView18 Recommendations Gillian Mcdermottasked a question related to Molecular MicrobiologyIs it possible to make up CHROM agar without bringing the solution to 100 degrees?Question5 answersJan 16, 2017I am trying to prepare agar plates from the CHROMagar powder and the instructions say to mix 33g in 1L of water and bring to the boil at 100 degrees before autoclaving. This is difficult for me as my lab does not have bunsen burners or waterbaths with this capability. I was wondering has anybody tried making this agar without boiling the solution before autoclaving. ThanksRelevant answerAlejandro MartinJan 16, 2017AnswerDo it on a microwave oven.Also, if your autoclave is an old model (where you can remove items from the sterilization chamber while the temperature is still relatively high) you might try autoclaving directly, without melting the agar beforehand, and then homogenizing the resulting solution afterwards.View12 Recommendations Ernest Obengasked a question related to Molecular MicrobiologyPlease is there an advantage of using agar well diffusion over agar disc diffusion in accessing antimicrobial effects of plant products?Question8 answersJan 12, 2017Want to know how efficient well diffusion is over disc diffusionRelevant answerAbdelahhad BarbourJan 15, 2017AnswerDisc or well diffusion tests are both qualitative assays. Hence, they wont give an accurate estimation of the effect of the antimicrobial activity of plants extracts or any other antimicrobial agent. Minimal Inhibitory Concentration (MIC) and IC50 values must be investigated for quantitative estimation. It all depends on what you are testing e.g. crude extract, protein extract, pure compounds or mixture of phenol-like compounds...doing well diffusion or disc diffusion test of crude or non-purified compound is meaningless... any plant extract would have some inhibitory activity due to the phenolic substances...  View10 Recommendations Rio Arnettasked a question related to Molecular MicrobiologyCan YCPlac vectors express genes in saccharomyces?Question2 answersJan 12, 2017Is ycplac33 not expressed in this saccharomyces without the introduction of other promoters?Relevant answerMarius K. SomdaJan 15, 2017AnswerDearMy contributionhttp://onlinelibrary.wiley.com/doi/10.1111/j.1567-1364.2011.00769.x/fullhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC55758/View0 Recommendations Syariffah Nuratiqah Syed Yaacobasked a question related to Molecular MicrobiologyWhat is the best addition media to culture lactic acid bacteria from honey?Question3 answersJan 12, 2017I have tried using MRS broth and plating on MRS agar with 0.01% calcium carbonate and there is bacteria colonies growth on plates. Unfortunately, almost all colonies shows catalase positive which is not belong to lactic acid family. Lactic acid bacteria is catalase negative or is it possible to have a catalase positive lactic acid bacteria?Relevant answerAbdelahhad BarbourJan 13, 2017AnswerLactic acid bacteria (LAB) are catalase-oxidase negative in general. MRS is usually used to cultivate LAB. In case you got other microorganism on your plate, it is highly suspected that you have contamination and in most cases this contamination is due to either Bacillus spp or other yeasts which can grow also on MRS. You may consider using M17 supplemented with 5% lactose or glucose. Do not use general media ! LAB needs enriched media to grow. In addition, most of G-negative bacteria can not grow on MRS. If you are not happy with MRS and M17, you can use Tryptic Soy broth supplemented with 2% yeast extract.Keep in mind that it is not as easy as it looks to isolated pure LAB from honey or any other fermented sources.-AbdelahhadView6 Recommendations Stephanie Morphetasked a question related to Molecular MicrobiologyDo I need to work aseptically with phage display to prevent contamination in my e. coli cultures when I amplify my phage?Question4 answersJan 12, 2017Hello everyone!I m starting a project involving phage display and the protocols largely sound like I can do my panning on the bench top, instead of in a biosafety cabinet.  Do I need to pan aseptically?  Or how do I prevent contamination downstream when I infect my E. coli cultures with the selected phages if the solution the phages are in is not sterile?  Do you filter the recovered phages before infecting?  Any suggestions?  General knowledge?Relevant answerWolfgang SchechingerJan 13, 2017AnswerStephanie, I think it largely depends on how clean (i.e. particle free) your lab environment is and what else is happening near and around you: Mold growing in the aircon and in/on fridges, colleagues happily eating crunchy food next to you and mice running around your bench (or your labemate s)?  In that case, (and in general, actually) better escape under a laminar flow, as the experiments are pretty lengthy and laborious, and the phage libraries are pretty precious and expensive resources.There is no need to panic about biosafety however, it s rather about protecting your samples and ensuring the quality of your data.Before you are going into the hot phase , the manual probably suggests to get used to the techniques involved and to do some phage amplification and retrieval and, possibly, playing around with a control phage panning experiment until you are sure you want to do the actual screening. So you actually may check safely if you don t get nasty contaminations.View0 Recommendations Angelbert Cortesasked a question related to Molecular MicrobiologyHi, do you have any idea on what research to do to test the bacterial community structure of the soil when you have a very low yield of DNA?Question19 answersJan 12, 2017I want to see the community structure of the soil amended with biochar, however, I ve got difficulty in extracting high DNA yield. My samples did not pass the quality control for next generation sequencing analysis, so I m thinking for another option with the same target yet with low conc. of DNA. Do you have any suggestions on what to do with my DNA with very low conc ( 5ng/ul but less than 15ng/ul) ? Thank you very much. Relevant answerHilary G MorrisonJan 12, 2017AnswerI take it you are sending the samples to a commercial facility with fixed standards for acceptable material? We sequence some extremely low biomass samples that come from extreme environments--some work, some don t.  If you take the risk that they won t work, would your facility make the attempt? Someone may suggest using whole genome amplification, but I don t advise it. Your results almost certainly will be biased. View5 Recommendations Samuel Majorasked a question related to Molecular MicrobiologyWill flowcytometry using a BD Accuri 6 FCM, be able to discriminate between SYBR-Green, Nile Red, and Chloroplast to count/ID bacteria and algae?Question2 answersJan 11, 2017I am going to run a flow cytometry experiment on synthetic communities of microalgae and bacteria. I would like to use a nuclear stain to determine all the biological particles, and gate those to ID microalgae Chloroplast fluorescence to ultimately determine the bacteria:Algae ratio. I will also be running a supplementary experiment to stain with Nile Red to reveal the effects on neutral lipid synthesis.The nuclear stain I think would be best suitable is SYBR-Green (or some kind of SYTO stain) because it should use the FL1 laser we have (ex laser: 488, FL1- 533/530). Nile Red should be picked up by the FL2 laser (ex laser: 488, FL2 - 585/40), and the Chloroplast should auto-fluoresce with the FL3 laser (ex laser 488, FL3 670 LP).Will this staining procedure/experiment achieve what I hope to? I should note I am a very amateur user of the flow cytometer and analyzing its data.Relevant answerAndrew Michael HoganJan 12, 2017AnswerHi Samuel, unless your lab uses Nile red frequently to assess lipid accumulation in multi-stain assays, I suggest avoiding it. Nile red exhibits solvatochromism in a hydrophobic environment (such as neutral lipid), and this causes a 40nm emissions to lower (more blue/green) wavelengths. As a result, it overlaps heavily with green fluorophores. I ve worked a lot with Nile red (but not with algae) and I know it will be detected in FL1 of your cytometer. Depending on how much lipid (hence staining) is present, you may or may not be able to compensate the signal from Nile red out of the SYTO signal. If you have the money for it, you can use other BODIPY or LipidTOX dyes that emit in the red and stain lipid.In terms of actually doing the experiment, Arno has good suggestions. To check for spectral overlap of the fluors, you can use samples stained individually with each fluor to set the compensation parameters (your flow technician should know how to do this).When you process your data, I suggest using FlowJo. You need to buy it , but its very good software. I havent used them, but free softwares are Flowing and CyFlogic.Hope this helps!Here s a paper for you on Nile red:https://www.ncbi.nlm.nih.gov/pubmed/4031658https://www.hindawi.com/journals/ijs/2016/5215086/cta/View0 Recommendations Ekaterina Avershinaasked a question related to Molecular MicrobiologyWhat is the efficient way of removing human dna sequencing reads? Are there any R-scripts available?Question14 answersJan 9, 2017I need to remove human dna data from HiSeq metagenome sequencing data of human gut. Is there any available script/software to use?Relevant answerCyril FrantzenJan 11, 2017AnswerHey Katya!I would recommend using the bbmap tool included in the BBTools suite for that task.For an introduction to the bbmap tool: http://seqanswers.com/forums/showthread.php?t=41057For a description of how to use bbmap to remove human contaminant DNA (also includes an already processed reference genome available for download):http://seqanswers.com/forums/showthread.php?t=42552View30 Recommendations Samira Fattahasked a question related to Molecular MicrobiologyWhat is the best way to store bacteria for short and long term?Question10 answersJan 10, 2017i need to know which method is more practical for storing bacteria with less change in genotype.Relevant answerTanny J K van der ReijdenJan 11, 2017AnswerMake a heavy suspension of your culture in glycerol broth (20% glycerol) and place the vial as soon as possible at -80°C. When needed scrape a little material from the surface of the froozen stock (prevend thawing of the stock) and culture in a suitable growth medium. Your strain will stay viable for many years this way. Using small srewcap vessels with one to one and a half milliliters glycerol broth will be enough.View8 Recommendations Fawzia Chaabane Chaouchasked a question related to Molecular MicrobiologyCan someone suggest me a protocol to evaluate antibiofilm activity?Question11 answersJan 2, 2017Hi,I want to evaluate the effect of supernatant from an actinobacterium on Staphylococcus aureus biofilm formation, but this supernatant showed antimicrobial activity against S. aureus, so i can’t evaluate the antibiofilm activity.  Please guide me how can i do this in simple way?Relevant answerMehdi Rostami RadJan 7, 2017AnswerTo evaluate the antibiofilm efficacy of different matherial such as titanium surfaces, the whole biomass present on each disc was detected. Briefly, biofilms grown on titaniumdiscs were air-dried and stained by disc immersion in a 5% crystal violet solution for 15 minute and, after several washing , air dried again. The estimation of biofilm biomass was performed by elution of the biofilm bound crystal violet with ethanol (96%)followed by the determination of the absorbance of 100 ll of eluted dye solution at 595 nm using a microplate photometer (Multiskan FC, Thermo Scientific; Milan, Italy). Measurements for each discs, were carried out in triplicate. For each bacteria andsurface, mean values and standard deviation of absorbance value were computed. Un-paired Student’s T test was used to compare data between experimental surfaces.I hope this protocol help youView12 Recommendations Paula Parreiraasked a question related to Molecular MicrobiologyHello everyone! Do you have any idea of companies that sell native Helicobacter pylori urease?Question2 answersJan 4, 2017 Native H. pylori Urease Relevant answerJai GhoshJan 6, 2017AnswerI do not know how far it be relied upon the supply of urease as mention by Mozafari. If I were you I would go in for isolation of the enzyme form the culture of H.pylori which would definitely very reliable for my research work. The isolation of the enzyme is pretty easy.View0 Recommendations Dagmar Stehlíkováasked a question related to Molecular MicrobiologyHave you got any advice of specifity loop-mediated isothermal amplification?Question4 answersDec 14, 2016Hi, I´m using this method for detection one of phytopathogenic bacteria. Now I´m testing new primers (without loop primers):reaction: 20 ul reaction with 2 ul 10x Isothermal Buffer (Optigene), 25mM MgSO4 change (3 - 5mM), 1.4 mM dNTP mix , 40uM of each FIP and BIP, 5 uM of each F3 and B3, and 8U GspSSD2.0 Polymerase (Optigene), 3,2 ul template DNA, and molecular grade water.I´ve had problem with contamination of negativ control (water). Now is good, but I have a problem with specifity and with some eror with preparation reactions.On two pictures is same reaction which is prepare with same procedure.from left: Xv - bacteria, which will detect, Xe,Ps,Ea - will no detect, NC - negative control, M - markerchange concetration of MgSO4 (from left 5mM, 4mM, 3 mM)Why I have got different results? (big mistake in preparation, beacause it is prepare to small amout reaction? bad designed primers? badly concetration MgSO4? Will I add betain?If anyone has faced the same problems and or know the cause and solution, I would really appreciate some help. Please email me at dagmarstehlik@gmail.com. Thanks! 16-12-06.jpg92.76 KB16-12-07.jpg142.72 KBRelevant answerRungrot CherdtrakulkiatJan 5, 2017AnswerFirst, I think it s good result for negative control. The Xv products are really good, but your problem is the false positive to other DNA, right? So, my comment is may be you can decreased the incubation time to be 30- 40 min because from you picture it s seem like the Xv product is very strong while the others are weak or low concentrations (except Ea and Cms). or may be you can increase temp. to be 66-67 C. PS. I have question about the concentration of the outer and inner primers in the reaction why you use the same concentrations (1.6 uM) or it is your mistake of typing information. Because normally the inner primers should be more than outer primers 8-10 fold. Good luck for you.View0 Recommendations Krishna K. Sharmaasked a question related to Molecular MicrobiologyCan someone suggest some experiments to depict the interaction of antimicrobial peptides on microbial cell membrane/surface ?Question6 answersJan 2, 2017For experimental evidences  for aggregation/ cell membrane penetration or self-assembling nature on surface or any other surface phenomenon by visualization techniques.Relevant answerBiswaranjan PradhanJan 3, 2017AnswerHi Krishna Kumar Sharma, I worked with a few antimicrobial peptides (Their role in immune modulation). Sometime during my study I planned to visualize the peptides in the cell membrane (or in cytoplasm), but dropped it because my goal is to see the immunological status of the mammalian macropage in the presence of the peptide. If you are trying to see the peptide aggregation on bacterial surfaces through florescence microscopy, label the peptide with Cy3 (Red) using protein labeling kit  (GE health care) and bacteria with CFDA/SE (Invitrogen) (Green) vital dye. Incubate the labeled bacteria and peptide together for 1 hour. Fix it with 2% para-formaldehyde and mount it on glass slide to see it under Confocal or florescence microscope. You can see the bacterial labeling with CFDA/SE here https://www.researchgate.net/publication/299576966_Comparative_Analysis_of_the_Effects_of_Two_Probiotic_Bacterial_Strains_on_Metabolism_and_Innate_Immunity_in_the_RAW_2647_Murine_Macrophage_Cell_Line. All the best for your research.Article Comparative Analysis of the Effects of Two Probiotic Bacteri...View14 Recommendations Erica Mangaraviteasked a question related to Molecular MicrobiologyIs double-band a problem before perform DGGE for ITS sequence?Question3 answersDec 22, 2016I am new in DGGE analysis. I am evaluating fungi profile from root samples and doing 2 PCRs with ITS primers, including the nested PCR, before run the DGGE. I wonder if two or three bands in an agarose gel are problematic in the DGGE analysis, or if it can be arbitrary, just related to the sample diversity. Is it possible to find it, or I d better avoid those samples?Thank you for attention!Relevant answerAnna Liza KretzschmarDec 23, 2016AnswerHi Erica,Could you include a picture of your gel to give us an idea how close together the bands are? Following on from Julian and Ali s questions, you need to work out if the multiple bands areunspecific or cross amplification - this will be bad for your analyses as these samples do not contain the sequences you wish to compare. Adjusting your PCR conditions or changing primers may help.ITS sequences of different length - if this is the case, there is already some selection going on between genera that fall into either of those sequence lengths. In this case I d recommend loading your entire PCR product on a gel, getting that resolution between the two (or three) ITS lengths then cutting out those bands. Those can then be purified using a gel purification kit (or simply  leaving the band in eppendorfs with molecular water in the fridge over night) and then load each band on a different lane in the DGGE.DGGE is a massive pain to work with. If there is any way you can change that part of your experimental design, I would highly recommend doing so.Also, one common problem with community analysis is that your answer is very dependent on the primer set you use. It s worth checking that the set you have selected is adequate at amplifying your target fungi so you don t unintentionally loose some information. Depending on what taxon resolution you re after, ITS may be too short a sequence to give you an ID down to species level when you get to the sequencing stage.All the best!View0 Recommendations Faïza Meriem Benabdounasked a question related to Molecular MicrobiologyCould anyone tell me if i can use Maldi-Tof MS for bacteria identification without using 16S rRNA gene sequencing?Question4 answersDec 20, 2016I want to identify my bacteria that i have isolated. I would also like to know the difference between the two techniques (Maldi-tof sequencing) in terms of results.Is it necessary to do biochemical tests, microscopic tests (gram test ...) before using the maldi-tof ms?Thanks,Meriem,Relevant answerFaïza Meriem BenabdounDec 23, 2016AnswerHi Alex,Thank you again for clarifying things.I will inquire about the database before starting to identify my bacterial strains.Have a good day,MeriemView0 Recommendations Caroline o carrollasked a question related to Molecular MicrobiologyHI, would trypticase soy yeast extract medium be ok to culture DSMZ bacteria strains (Strep.uberis, Strep.agalactiae , S.aureus) ?Question5 answersDec 19, 2016I am going to be growing the three mention strains and just wondering if trypticase soy yeast extract medium be ok for all three strains or do i need to grow them on more selective media ?Relevant answerCaroline o carrollDec 21, 2016AnswerHi JorgeThank you so much for your help. Just a few more things, should i do an OD before i plate them or just plate them after 24hrs ? Also should I test the them on selective agar just to make sure the bacteria is the strain i want ?View4 Recommendations Nicholas Carmodyasked a question related to Molecular MicrobiologyPseudoalteromonas citrea growth conditions?Question2 answersDec 19, 2016I have started work on culturing a strain of bacteria, Pseudoalteromonas citrea, I have read into the background of growing the strain but I would like to know if anyone has dealt with this bacteria in the past and if so, what growth conditions were used. Any information will be greatly appreciated.Relevant answerNicholas CarmodyDec 20, 2016AnswerThank you for your response, I have used the aforementioned Difco marine agar and those plates appear to stimulating better growth, I have also noticed an increase in colonies by changing the incubation temperature to 28 degrees.View0 Recommendations Sim Huey Yiasked a question related to Molecular MicrobiologyCan anyone suggest a quick and easy way of the purification of fungus DNA?Question5 answersDec 16, 2016I do not have any kit to be use in purification. Can anyone suggest an effective and easy way to purify the DNA? Thank you in advanced! Relevant answerBradley MitchellDec 20, 2016AnswerSim Huey Yi:The following reference may help you.  The full article should be available through open access.Girardin H, Latge JP, Srikantha T, Morrow B, Soll DR. Development of DNA probes for fingerprinting Aspergillus fumigatus. J Clin Microbiol. 1993;31:1547–1554.View0 Recommendations Peter Balázs Kósasked a question related to Molecular MicrobiologyDoes anyone have the sequence of pRK2013 or DSM-5599 or PC-V3125 plasmid?Question3 answersDec 15, 2016I d like to check the identity/integrity/validity of the plasmid with the alternative names above. The size is too large for a simple gel, so restriction fragments should rather be inspected, but for that I d need to know the sequence to see if the pattern is correct.I could not find the sequence in NCBI - Addgene - DSM - Stanford - etc... :(So we ll blindly use it and if it works, then fine. :)Relevant answerPeter Balázs KósDec 20, 2016AnswerThanks a lot.View0 Recommendations Muhammad Aslamasked a question related to Molecular MicrobiologyI was trying to isolate Lactobacillus DNA through boiling method, but got fragmented band. Why is it so, anyone kindly guide me?Question5 answersDec 15, 2016I was trying to recover the Lactobacillus DNA through simple boiling method. Some of the samples failed but one showed fragmented band. The boiling method include:Loopful colonies from MRS media. Washed with PBS. Heat it at 99C, centrifuged it and ran gel electrophoresis. Relevant answerMuhammad AslamDec 19, 2016AnswerThank you @Ali Mahmoudpour for sharing your experience. View4 Recommendations Caroline o carrollasked a question related to Molecular MicrobiologyHi, does anyone have experiences in co-culturing cell lines (MAC-T ) with bacteria (E.coli , S.aureus , strep. uberis )?Question2 answersDec 16, 2016I am relatively new to cell culturing. I need to co - culturing MAC-T cell with bacteria. If anyone would have a protocol or any papers that might be relevant , I would greatly appreciate it .Relevant answerBiswaranjan PradhanDec 16, 2016AnswerHi Caroline,I co-cultured Raw264.7, THP-1 and HCT-116 cell lines with Lactobacillus acidophilus, Bacillus clausii, Bifidobacterium bifidum and Saccharomyceses cereviseae to find out their role in activating the host innate immunity and with Salmonella typhimurium to find out its cytotoxicity and inflammatory property on macrophage. You can find the paper here (http://link.springer.com/article/10.1007/s12602-016-9211-4).View5 Recommendations Dong Yangasked a question related to Molecular MicrobiologyWhat is the composition of the capture buffer when purify DNA from enzyme reaction or gel band?Question2 answersDec 14, 2016i want purify DNA from enzyme reaction, how to prepare the capture buffer with guanidine?Relevant answerHoward JuncaDec 16, 2016AnswerYou need to use a solution with very high concentration of guanidinium thiocyanate or guanidinium HCl, as Silica beads or columns (filters) are pretreated with strong acids to activate that capture of nucleis acids, and later on is important to keep in washing solutions very high salt and alcohol concentrations to avoid release. Some hints, working solutions and trials at:  https://arbol.uniandes.edu.co/Archivos/Ivanova-Membrane-Based%20Protocol.pdfView4 Recommendations George Flemingasked a question related to Molecular MicrobiologyHello I would like to know if anyone knows of a way I can do a bacterial assay that requires acidic medium?Question6 answersDec 12, 2016I have developed polymer surfaces that release bactericidal agents at pH 4. I am currently initiating the release by placing the surfaces in acetate buffer (pH 4) for 30 s, before transferring the sample into a bacterial solution, in a pH 7 nutrient broth. There is no kill being observed and I think this is due to the fact that ideally the sample needs to remain in acidic environment throughout the duration of the incubation. Does anyone know of any bacterial assays where I can keep the pH low throughout?Thank youRelevant answerPiyush Kumar TiwariDec 15, 2016AnswerDear George,I am agree with Traci, so please go with the acid tolerating bacteria like lacobacillus or Streptococcus thermophilus. But for bactericidal point of view you should customize your polymer so that it could have broad range activity and can transform into potential commercial product. Best RegardsPiyushView4 Recommendations Sreeram Chandra Murthy Peelaasked a question related to Molecular MicrobiologyHow to perform in silico analysis of an enzyme s action on gram positive cell wall?Question1 answerDec 12, 2016Hi,I wish to apply in silico methods for analysing the activity of a protein. the protein is actually an enzyme that destroys gram positive cell walls. I wish to model the effect of various ligands on the activity of the enzyme. However, there is no structure of gram positive cell walls in any database. Previous publication used N-acetyl glucosamine - pentapeptide for their in silico analysis. I couldn t find any structure file for the same. Please tell me how can I create a structure of N- acetyl glucosamine - pentapeptide for insilco analysis? Thank you.Relevant answerJonathan Malcolm FriedmanDec 13, 2016AnswerIt all depends on what software you have available.  I tend to use the old graphics program O from Uppsala Software Factory .  The PDB (www.rcsb.org) has several N-acetylglucosamine containing ligands bound to proteins that you can find by Advanced Search at the PDB site using the Chemical components / ligands key (search: N-acetylglucosamine) though as you say, none of the ones have NAG  attached to a peptide.  For starters, you can just find the files containing NAG ligands and editing /copying the appropriate atoms from the text file leaving out the unnecessary atoms.  For the peptide, the graphics program O has a way to go from sequence to a chain, but at this point, there are enough protein structures in the PDB that you can also probably find something close to your pentapeptide in an existing structure.  First do a BLAST search to find proteins that may contain a similar peptide; find an appropriate PDB file for the BLAST hit; and then extract the peptide by editing it from of the PDB text file.  Then you can use whatever graphics software to get the NAG and peptide close enough and at about the right orientation to start fixing it up with appropriate energetics software.  If you ever have a new ligand there are ways to go from a ChemDraw Drawing to coordinate files.  (e.g. Copy as SMILES option allows you to paste into a .smi text file and there is software available to convert the SMI file to a pdb coordinate file:  Google search smi to pdb)  It all depends if you have ChemDraw or some other graphics program and molecular energetics/mechanics programs to fix-up the initial structures.  Some advanced versions of ChemDraw also give you PDB files directly.View0 Recommendations Angelbert Cortesasked a question related to Molecular MicrobiologyIs it possible to use Dobereiner s medium to count the total population of Nitrogen-fixing bacteria in the soil?Question5 answersDec 6, 2016I m wondering if all those capable of fixing nitrogen can grow in a nitrogen-free medium, Dobereiner s medium in particular. I am considering the media might not allow the growth of other nitrogen fixers due to differences in optimal growth condition requirements. Thank you.Relevant answerValter TandoiDec 12, 2016AnswerWhenever you wish to describe a microbial population composition by cultivation you always have the  bias that some relevant clusters did not grow (a medium that allow the growth of all biomass components does not exists). To avoid doubts the only  way is to combine the traditional cultivation approach (for me anytime fundamental) with a modern biomolecular approach: PCR and  FISH (Fluorescent in situ hybridization). What in my opinion is fundamental, is to test anytime the results of PCR on the original sample (methods are available to extract bacteria from soil and transfer them in a state suitable for FISH). FISH will confirm PCR results.View5 Recommendations R.A Tan Chopsasked a question related to Molecular MicrobiologyWhat is the rationale of using skimmed milk in protease activity and elastase activity of pseudomonas aeruginosa?Question8 answersNov 30, 2015on the Effects of Antibiotics on Quorum Sensing in Pseudomonas aeruginosa paper by skindersoe et. al, they have used skimmed milk.i am searching for a test for protease and elastase. im going to use a plant extract of tannins, is it possible that i use that method to?how can that test (skim milk test)  prove the effectivity of my sample? or how what is the manifestation of a postive result for this test?Relevant answerAlaa Jabbar Abd Al-ManhelDec 11, 2016AnswerI agree with all answers above ...good luck.View8 Recommendations Reecha Sahuasked a question related to Molecular MicrobiologyWhat should be the right way to calculate the enzyme activity of microbial lipase using p-NPP as a substrate?Question12 answersDec 5, 2016reaction condition:concentration of substrate is 800 µMenzyme added is 250 µLtotal reaction volume is 1.5 mLand the amount of p-nitrophenol released from the substrate is 4 µg/mL in 30 minutes.I have not calculated the amount of protein in 250 µL of my enzyme.Relevant answerDominique LigerDec 9, 2016Answer@ Ewa:I m afraid you are mixing two essential notions in enzymology:steady state condition which is conditioned by [S]0 [E]0 and [P] formed during the laps of time considered negligible compared to [S]0 (so you see it has nothing to do with Km value). This is required to measure initial velocity v0.and enzyme saturation which implies a further condition where Km appears: [S]0 Km.  This is required to observe v0=vm=kcat[E]0If the steady state condition is respected then Michaelis Menten equation applies whatever [S]0 being far greater than [E]0(including [S]0 Km):v0=kcat[E]0x([S]0/(Km+[S]0)). So it means that for whatever [S]0 being far more than [E]0, one can define a laps of time where this equation applies (ie. velocity is constant so P formed versus time is linear), OK? If the laps of time where the steady state applies is very small though then one way to enlarge the laps of time where equation applies is to use less enzyme to decrease v0 ( as v0 depends linearly on enzyme concentration) , OK? If as Reecha made, the time point considered is beyond the linearity limit then one way to get accurate v0 determination is either to shorten the time of reaction or to diminish the enzyme concentration.In the special case when [S]0 Km but still far more than [E]0 then:v0=(kcat/Km)[E]0[S]0 and equation is still about initial velocity. So this equation applies as long as [P] formed remains negligible compared to [S]0 and  when P is formed linearly versus time.The only difference here is actually that the reaction rate is order 1 towards both E and S where it is 1 only towards E in the general equation. One way to slow down the reaction for a fixed [S]0 would also be to use less enzyme in order to have a better determination of v0.Hoping it helps to make things clearer...View5 Recommendations Muhammad Fazle Rabbeeasked a question related to Molecular MicrobiologyWhat is the best method for antibacterial activity assay of endophytic bacteria?Question4 answersDec 5, 2016RabbeeRelevant answerKasi GopinathDec 6, 2016Answerwell diffusion methodView12 Recommendations Debashis Halderasked a question related to Molecular MicrobiologyCan anyone provide me the most efficient protocol for isolation of bacteriocin form lactobacilli/lactic acid bacteria?Question6 answersDec 6, 2016I would also like to known, what % of gel I should use in SDS-PAGE to find out the proper band of bacteriocin. Whether any specification for staining and De-staining...Thanks in advance.Relevant answerAnila KumariDec 6, 2016AnswerBacteriocins are low molecular weight peptides.Best method to purify bacteriocins from LAB is to use Tricine-SDS PAGE with upto 15% polyacrylamide gel concentration.hope you will get desired resultsgudluck!!View5 Recommendations Mahboubi Mohaddeseasked a question related to Molecular MicrobiologyQuestion about ligation?Question3 answersDec 5, 2016DearI prepared my cloning reactions with positive and negative controls of my reaction. The positive control was pET with double digestion in presence of T4 DNA ligase and the negative Control was double digested pET  without T4 DNA ligase. I electrophysed the reactions by agarose gel. I can see the DNA fragments in negative control on the gel but I can not see the related bonds after incubation for ligation reaction and positive control. I want to know is it logical and how we explain this? ThanksRelevant answerTausif AlamDec 6, 2016AnswerYes, what you see is an expected result. Normally, the recommended conditions for ligation include considerable excess of the enzyme and you will see large polymers. If you really wish to a single event, used far less ligase.View0 Recommendations Anna Griegoasked a question related to Molecular MicrobiologyHartmans de Bond medium for Mycobacteria. Is it possible to find a commercial solution for it?Question4 answersDec 4, 2016Hello!So I looking how to induce starvation in M.smegmatis and I found an article that used the minimal Hartmans de Bond medium, where the source of carbon, nitrogen and phosphate were really reduced. So I decided to use this medium. Have someone used it? Is it possible to find a commercial solution for it? Its preparation is really long and could introduce a lot o variableThank youRelevant answerVincent Olivier BaronDec 5, 2016AnswerHi,I could not find any commercially available Hartman s de Bond medium online. We often come accros modified harman s de bond medium in the literature for example: (2004, Shleeva et.al Formation of ‘non-culturable’ cells of Mycobacterium smegmatis in stationary phase in response to growth under suboptimal conditions and their Rpf-mediated resuscitation which show another protocol to prepare the medium.Another possibility could be to used 10% Dubos medium as a starvation medium. this is what have been done in the multiple stress model of Deb et al. 2009 ( Deb C, Lee C-M, Dubey VS, Daniel J, Abomoelak B, Sirakova TD, et al. A novel in vitro multiple-stress dormancy model for Mycobacterium tuberculosis generates a lipid-loaded, drug-tolerant, dormant pathogen. PLoS ONE nd;4 ). you can buy Dubos medium online.I hope this help, good luck!best wishes,Vincent. View10 Recommendations Anil Yousafasked a question related to Molecular MicrobiologyHow to get more extracellular methanol induced protein expression in Pichia X-33?Question7 answersNov 29, 2016I am expressing 24KD protein in pichia X-33 under alcohol oxidadase AOX promoter. I transformed linearized plasmid with electrotransformation, then i screened clones on high concentration of zeocin 200ug to 1500ug. I did colony PCR then the best clones were picked and expression was performed BMMY expression media with 1% methanol, but i got very little or no expression. please suggest me something to get more expression. Relevant answerTom MasiNov 29, 2016AnswerAre you using the intracellular or secreted pathway for expression? What is the density of the cells prior to induction? How long did you induce expression for? Did you do a time course (24, 48, 72, 96 hour) to determine if and when expression switches on/off? View6 Recommendations Büşra Aktaşasked a question related to Molecular MicrobiologyIs it possible the classification antibiotic resistance genes?Question5 answersNov 28, 2016As we know antibiotiic resistance is a dire clinical problem with important ecological dimensions. It is in human pathogens continues to rise at alarming rates.I am searching for antibiotic resistance genes classification systems. Is it possible to classificate them. If it is possible, which way should i choice or which algorithm should i use for ?Relevant answerJose Sergio HleapNov 29, 2016AnswerHi, I am not sure about classification systems. My guess is that it would be a very hard task, given that most of the antibiotic resistant strains come from mutations (often point mutations) in regular genes. However, there are databases that you can query about known antibiotic resistance genes, and therefore you can access to annotated information of such genes. Some of these resources:https://ardb.cbcb.umd.edu/https://card.mcmaster.ca/: This one has a software to try and classify your sequencesThere are also tools developed that query more than one database. One has been described in https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910750/.Hope this is of help,SergioView8 Recommendations Andrew N Galeasked a question related to Molecular MicrobiologyQuestion on whether a sample of DNA is good enough for shearing using a bioruptor?Question5 answersNov 28, 2016I am currently trying to prepare a library for illumine deep sequening using the yeast Candida glabrata. Ive been having difficulty generating a high quality library. My collaborators believe that it is my genomic extraction that is causing the problem, with some protein or some leftover buffer from a previous step. I was using a modified version of the master pure yeast DNA extraction kit with a phenol chlor extraction at the end. Whenever I would go to sonicate my DNA I would have a large amount of fragments that were of low MW (less than 50 bp according to the BioA) a low amount of normal fragmentation (between 250 and 400 bp) and a large amount above 700 or so bp, with the lower than 50 bp outweighing the others by far in terms of molarity and weight. As such they recommended that I use a new extraction protocol and do ti alongside someone in their lab. Based on their recommendation I am currently trying the ZR fungal/bacterial genomic mini prep kit. Me and another ran the protocol using a highly saturated glycerol stock. Their prep had great fragmentation mine looked like a lot of protein contamination. When i nano dropped the samples the one that worked had a 260/280 of 2.0 and a 260/230 of .7. The one that did not work had a 260/280 of 1.6 and a 260/230 of .35Based on the fragmentation (too much in higher MW fragments) and the low 260/280 I believe that the fragmentation for the second one failed due to protein contamination. So i started taking to zymo and they gave me some recommendations. After following them I got samples with a 260/280 of 1.85 and a 260/230 of .~1.9-2.0. So based solely on the nano drop these samples seem much better. (they also have a higher yield). However when I run them on a gel I get a very concerning smear. The first gel image includes the original attempt, in which one had good fragmentation the other bad fragmentation. It is a 1kb NEB ladder. The third well with sample was the extraction that had good fragmentation, the fifth well with sample had the bad fragmentation.  The second image contains my most recent attempts with all but the first 2 sample containing wells coming from the extraction kit. It looks like there are fragments in this smear from 10kb down to 1kb if not lower. However if I am looking at the first one correctly it seems that the one that had good fragmentation also had a smear that went to low MW. It may just not be as visible due to the large difference in initial loading concentrations. My question to everyone is whether or not they think any of the 4 samples in the second gel image would be acceptable to carry over for fragmentation and subsequent library preparation?Thank you for your time Image90.jpg28.03 KBImage92.jpg23.38 KBRelevant answerAndrew N GaleNov 28, 2016AnswerDoes the second protein precipitation step greatly increase the purity of the sample? View0 Recommendations Guangshan Weiasked a question related to Molecular MicrobiologyHow to do a scientific sampling when we perform a microbial diversity investigation on natural environments, such as soil, seawater etc.?Question5 answersNov 21, 2016There are so many microbial diversity investigations using high-throughput sequencing on variety of natural environments, e.g. land soil, fresh and salt water, sediment etc. Some of them sampling their samples in a site, and then mixed them for one sample to do the next study. I want to know if this kind of sampling method are scientific and rigorous? And how to do a scientific and rigorous sampling when we do microbial diversity investigation in natural environments?Thank you!Relevant answerChristopher M M FrancoNov 27, 2016AnswerHi WeiYour sampling should reflect the research question you want to ask,  As you know, soil is full of microsites, each with is own microbial diversity. So depending on what you wish to know you have to sample accordingly. It is alo necessary to validate your experimental methods to ensure that they are giving you a true representation.Best wishesChrisView6 Recommendations Eli Manfredasked a question related to Molecular MicrobiologyCan any one differentiate the condition below?Question4 answersNov 20, 2016Why there is bacterial growth on LB agar with and without ampicillin when plated with competent cell? But, no bacterial growth on both LB agar with and without ampicillin when plated with transformantRelevant answerManjunatha KogenaruNov 21, 2016AnswerPossibly, your transformation process is killing the bacteria hence no transformants even on the LB agar plate without antibiotic.   All the best,ManjunathaView6 Recommendations Marzieh Sanaeiasked a question related to Molecular MicrobiologyWhy after 9 rounds of cell selex I don t have any increase in FITC intensity?Question1 answerNov 16, 2016I do cell-selex on candida albicans.after 9 rounds of selex,flowcytometery shows no increase in FITC intensity from 1 to 9 round.Relevant answerJai GhoshNov 18, 2016Answer( round is too small a figure. To get a significant increase in FITC you need atleast 50 to 100 rounds. Flowcytometers are not so sensitive as you are expecting.View0 Recommendations Matthew Stablerasked a question related to Molecular MicrobiologyAfter extracting the DNA from bacteria into a DNA extraction plate what would be the best way to safely dry the DNA to the wells of the plate?Question4 answersNov 17, 2016I was thinking either ethanol or isopropyl alcohol precipitation would work but I can t seem to locate a good protocol for doing this. I need the wells to be dry because the plates I m working with are going to be shipped out from my company to others. I do not want to risk shipping the plates with liquid in the wells because of possible cross contamination. Also, what would be the best buffer/media for the person receiving the dried DNA plate to use for rehydrating the DNA in the wells, would TE buffer be the best option? It would be greatly appreciated if someone could point me in the right direction to where I could find a good protocol for doing this...if there is one. Hopefully, I worded this ok! Thanks in advance!Relevant answerOsric Rahul PatharkarNov 17, 2016AnswerIsopropanol precipitation would certainly work. However, simply heating your plate at 55-65 degrees Celsius (in an incubator) until dry would be the easiest. Re-hydration could be with water or TE.For isopropanol precipitation: add sodium acetate to 0.3 M and add 0.6 volumes of isopropanol. Mix. Spin =5000xg for 10 minutes and decant the supernatant. Wash the pellets once with 70% ethanol and allow to dry.View5 Recommendations Paul Riosasked a question related to Molecular MicrobiologyCalculate a colony count?Question13 answersNov 10, 2016For example, if I put 30uL of a bacteria suspension(dil 10-3) and I count 25 colonies at a agar plate. The CFU/mL will be (25x1000/30)x103? Is that ok?Relevant answerAlaa Hani Al-CharrakhNov 14, 2016AnswerHi:Your calculation is right.  Don t be afraid; Go ahead sir.View28 Recommendations Abdelkader Senoussi Elzenasked a question related to Molecular MicrobiologyBest culture media for gliding bacteria?Question2 answersNov 7, 2016I m looking for the suitable media for growth gliding bacteria (articles for myxococcus xanthus bacteria)?   Relevant answerOlivier BraissantNov 14, 2016Answera good source of information can be found in the handbook of microbiological media (just google it). The most difficult will probably to choose one from the long list of possibilities that are provided in this book.View5 Recommendations Basant Aliasked a question related to Molecular MicrobiologyWhen do e.coli BL21 cells secrete beta-lactamase?Question2 answersNov 10, 2016How long will the e.coli cells need to secrete beta-lactamase?Relevant answerMarius K. SomdaNov 14, 2016AnswerDear BasantSecretion of  β-lactamases (MβLs) are zinc-dependent enzymes produce β-lactamases  are synthesized in the bacterial cytoplasm as precursors and are secreted into the periplasm. These enzymes are secreted by bacteria to  hydrolyze most β-lactam antibiotics and to resistant to all clinically employed inhibitors.RegardsMariusView0 Recommendations Sudhanthiramani Sudhanthirakodiasked a question related to Molecular MicrobiologyCan anyone please explain how to differentiate the origin of any organism by molecular techniques?Question8 answersFeb 9, 2016Hello everyone,I want to find out the origin of microorganisms whether its from human or animals or environment by using advanced molecular techniques. If any one knows the techniques please let me know. It will help me in doing research on antibiotic resistant bacteria. with advance thanks,Dr.S.SudhanthiramaniRelevant answerAlaa Hani Al-CharrakhNov 13, 2016AnswerHi:There are so many tools for comparison of nucleotide sequence of different species using the science of Bioinformatics . One of these tools is by doing sequence homology via Nucleotide BLAST and by Phylogenetic tree.Best  regardsView21 Recommendations1234AdvertisementJoin ResearchGate to find the people and research you need to help your work.20+ million members135+ million publications700k+ research projectsJoin for freeSimilar topicsMicrobiology TechniquesMicrobial BiotechnologyMicrobial Molecular BiologyPCRMolecular Biological TechniquesMicrobial EcologyApplied MicrobiologyMolecular BiotechnologyAntibiotic ResistanceEnvironmental MicrobiologyHighly-cited researchers Jos VanderleydenKU Leuven Hedvig Engström JakobssonSahlgrenska University Hospital Torben Lund SkovhusVIA University College Nicolas BarraudInstitut Pasteur Liisa KauttoMacquarie University James C PatonUniversity of Adelaide Mary P CarrollUniversity Hospital Southampton NHS Foundation Trust Leo SchoulsNational Institute for Public Health and the Environment (RIVM) Gavin CollinsNational University of Ireland, Galway Laura Espinosa AsuarUniversidad Nacional Autónoma de México Nobuhiro TakahashiTohoku University Alex MiraFISABIO Seana K DavidsonIEH Laboratories and Consulting Group Yurong WenXi an Jiaotong University Richard B. EckertDet Norske Veritas Jonathan Kirk HarrisUniversity of Colorado Théodore BouchezFrench National Institute for Agriculture, Food, and Environment (INRAE) Lisa BerryUniversity Hospitals Coventry and Warwickshire NHS Trust Suzannah M Schmidt-MalanMayo Foundation for Medical Education and Research Aura Ontiveros-ValenciaInstituto Potosino de Investigación Científica y TecnológicaDidn t find what you re looking for?Search for more research, methods, and experts in other areas on ResearchGate.Discover more researchAdvertisement orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleWelcome back! Please log in.Email · HintTip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleNo account? 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