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Storeat4°C.Adjustment:Beforeuseaddbeta-mercaptoethanolto0.15%. Modeofaction:Abacteriostaticagentthatinterfereswithbacterialproteinsynthesisbybindingtothe50Ssubunitofribosomesandpreventingpeptidebondformation. Mechanismofresistance:Theresistancegenespecifiesanacetylytransferasethatacetylatesandtherebyactivatestheantibiotic. Stocksolution:50mg/mlin100%ethanol.Storeat-20°C. pBeloBAC11plasmidpreparation:30ug/ml BACtransformation&minipreps:12.5ug/ml Stocksolution:200mg/mlinH2O.Filtersterilizeandstoreat-20°C. BACtransformationplates:240ml/plate(15X150mm) SOLUTIONSFORBACLIBRARYCONSTRUCTION
10XHomogenizationBuffer(HB)stock:(1liter)
Ingredient Amount FinalConcentration Trismabase 12.1g 0.1M KCl 59.7g 0.8M EDTA 37.2g 0.1M Spermidine-HCL 2.55g 10mM Spermine-HCL 3.48g 10mM Adjustment:pHto9.4-9.5withNaOH.Storeat4°C.
1XHomogenizationBuffer(HB):(1liter)
Ingredient Amount FinalConcentration 10XHBstock 100ml 1X Sucrose 171.15g 0.5M Storeat4°C.Adjustment:Beforeuseaddbeta-mercaptoethanolto0.15%.
20%Triton-X100in1XHB:(100ml)
Ingredient Amount FinalConcentration 10XHBstock 10ml 1X Sucrose 17.12g 0.5M TritonX-100 20ml 20% Storeat4°C.
WashBuffer(1XHomogenizationBuffer(HB)plus0.5%TritonX-100):(1liter)
Ingredient Amount FinalConcentration 10XHBstock 100ml 1X Sucrose 171.15g 0.5M TritonX-100(20%stockin1XHB) 25ml 0.5% Nalaurylsarcosine(2%):(1liter)
Adjustment:AutoclaveIngredient Amount FinalConcentration Nalaurylsarcosine 20g 2% LysisBuffer:(1liter)
Adjustment:Beforeuse,addProteinaseKto0.1mg/mlIngredient Amount FinalConcentration EDTA 500mlof1Mstock 0.5M(pH9.0-9.3) Nalaurylsarcosine 500mlof2%stock 1%< EDTA(0.5M,pH8.0):(1liter)
Adjustment:pHto8.0withNaOHorHCl.Autoclave.Ingredient Amount FinalConcentration EDTA 186.1g 0.5M NaOH 20g EDTA(1.0M,pH9.0to9.3):(1liter)
Adjustment:pHto9.0to9.3withNaOH.Autoclave.Ingredient Amount FinalConcentratio EDTA-disodiumsalt 362.2g 1.0M NaOH 40g Tris-HCl(pH8.0):(1liter)
Adjustment:pHto8.0withHClandadjustvolumeto1liter.Autoclave.Ingredient Amount FinalConcentration Trismabase 121.1g 1M HCl 42ml ddH20 800ml T10E1(pH8.0):(1liter)
Adjustment:AutoclaveIngredient Amount FinalConcentration Tris-HCl 10mlof1Mstock,pH8.0 10mM EDTA 2mlof0.5Mstock,pH8.0 1.0mM PMSFphenylmethylsulfonylfluoride
Caution:PMSFisahighlytoxicacetylcholinesteraseinhibitor.ToavoidpossIBLelabcontamination,wepurchasesmall250mgaliquotsandthenaddisopropanolasabovetotheentirebottletomakeafinalconcentrationof100mM.
Storeat4°C.Ingredient Amount FinalConcentration PMSF 250mgvial 100mM Isopropanol 14.35ml SOCMedium(forE.colitransformation):(1liter)
Adjustment:AdjustpHto7.0,autoclave,andcooltoroomtemperatureandthenadd.Ingredient Amount FinalConcentration Bactotryptone 20g 2% Bactoyeastextract 5g 0.5% NaCl 10ml(1Mstock) 10mM KCl 2.5ml(1Mstock) 2.5mM ddH2O upto980ml -
a:Prepareandfilter-sterilizea2MMg++stock(1MMgCl26H2O+1MMgSO4-7H2O)b:Prepareandfilter-sterilizea2MGlucosestock.MgSO4/MgCl2Stocka 10ml(2Mstock) 20mM Glucoseb 10ml(2Mstock) 20mM LBMedium(E.coli):(1liter)
Adjustment:AdjustpHto7.5.Autoclave.Ingredient Amount FinalConcentration Bactotryptone 10g 1% Bactoyeastextract 5g 0.5% NaCl 10g 1% Agar(forplates) 15g 1.5% Lysozymesolution:(100ml)
Adjustment:Adjustvolumeto100mlwithpurewaterandautoclave.Storeat4°C.Addlysozymejustbeforeuse.Ingredient Amount FinalConcentration Glucose 0.9g 50mM EDTA(pH8.0) 2mlof0.5Mstock 10mM Tris-HCl(pH8.0) 5mlof1Mstock 25mM Lysozyme 5mg/ml NaOH-SDSstock
Adjustment:MixNaOHandSDSstocksjustpriortouse.Ingredient Amount FinalConcentration NaOH 1/2volumeof0.4Nstock(8g/500ml) 0.2N SDS 1/2volumeof2%stock(10g/500ml) 1% Potassiumacetatestock(pH4.8-5.3):(100ml)
Ingredient Amount FinalConcentration PotassiumAcetate 60mlof5MKOAc GlacialAceticAcid 28.5ml H2O 11.5ml 20XSSC(pH7.0):(1liter)
Adjustment:DissolveinpurewaterandadjustpHto7.0withCitricacid.Ingredient Amount FinalConcentration NaCl 175.3g/l 3.0M NaCitrate 88.2g/l 3.3M 50XTAE(ElectrophoresisBuffer):(1liter)
Dilute50foldwithpureautoclavedwater.Ingredient Amount FinalConcentration TrismaBase 242g Glacialaceticacid 57.1ml 2.5MTris-Acetate EDTA 100mlof0.5MEDTA(pH8.0) 50mM 5XTBE(Electrophoresisbuffer):(1liter)
ThepHshouldbeabout8.3andshouldnotrequireadjustment.Ingredient Amount FinalConcentration TrismaBase 54g 0.45M BoricAcid 27.5g 0.45M EDTA 20ml(0.5Mstock) 10mM Chloramphenicol(CM)
Isopropylthiogalactoside(IPTG)
5-bromo-4-chloro-3-indolyl-beta-D-galactoside(x-Gal)
Stocksolution:20mg/mlinNN-dimethylformamide.Storeat-20°C.
10XgelloADIngbuffer:500ml
Ingredient Amount FinalConcentration Glycerol 350ml 35% 5XTBE 10ml 1X 0.5MEDTA(pH8.0) 20ml 20mM 20%SDS 5ml 0.2% 10%bromophenolblue 30ml 0.6% ddH2O 85ml -
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