Deprecated: Required parameter $cat_id follows optional parameter $type in /www/wwwroot/ebimall.com/systems/hong.php on line 2088

Deprecated: Required parameter $where follows optional parameter $tree_id in /www/wwwroot/ebimall.com/systems/hlb.php on line 3505
Calculating Concentrations for PCR188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
您好,欢迎您进入188进口试剂采购网网站! 服务热线:4000-520-616
蚂蚁淘商城 | 现货促销 | 科研狗 | 生物在线

Calculating Concentrations for PCR

i)Oligonucleotideprimersaregenerallysuppliedas"somanyODunits/ml"-butwhatdoesthismean,intermsofmg/ml,ormmol/ml,etc?

Given:aprimerisYnucleotides(nt)long;

Given:theMWofssDNAis(330daltonspernt)x(lengthinnt)(Sambrooketal.,1989;p.C.1);

Given:theconcentrationofprimer(=ssDNA)producinganODof1at254nmina1cmcuvette,is37ug/ml;

Then:theMWoftheprimeris330.Ydaltons

And:XOD/ml=37.Xug/ml=37.Xmg/l=37.X/330.YmM=37.X.1000/330.YuM

Forexample:

B88/77primer-a17-meroligodeoxynucleotide-assuppliedis12.6ODunits/ml.Weneedtomakea5uMstocksolutionforPCR.

MW:17x330=5610

Concentration:12.6ODx37ug/ml=466ug/ml=466mg/l=0.466g/l

Molarity:0.466/5610=0.000083Molar=83uM

Therefore:weneed5ulofoligostocksolutionin83ul(+78ulwater)tomakea5uMsolution(if1ulin83ulgivesa1uMsoln...)

ii)CalculationofamountsforPCRreactions:ifweneedafinalconcentrationof0.5uMoligointhePCRreactionmix(finalvolume50ul),weadd5ulof5uMstocktothereactionmix(1/10finaldilution).

StocksofnucleotidesforPCR(orotherprocedure)areNEARLYALWAYSdNTPs(deoxynucleotides),andconcentrationsisalmostalwaysgiveninEACHdNTP:thatis,thegivenconcentrationisEACHnucleotideinthemix,NOTthetotalconcentration.Thismeansthata2.5mMdNTPmixforPCRcontains2.5mMofEACHdNTP,and10mMTOTALdNTPs.

Example:

i)Makeupa2.5mMstocksolutionofdNTPsfromstock100mMindividualdNTPs,suppliedbyPromega:

  • FIRSTmixequalvolumesofeachnucleotide(eg:50ul):thisgivesyou200ulof25mMmixeddNTPs(Remember:concn.expressedinEACHdNTP).
  • THENdilutethis(oraliquot)1/10withWATER-aliquotinto100ulamountsandfreeze.

ii)Preparea1mMstockofdNTPswithdTTPsubstitutedto10%(w/w)bydigoxigenin-11-dUTP(DIG-dUTP)foruseasalabellingmixforPCRlabellingofPCRproducts:

GIVEN:

  • DIG-dUTPsupplied(byBoehringerMannheim)at25nmol/25ul=1umol/ml=1mM;finalconcentrationofDIG-dUTPmustbe1/10ththatofothernucleotides,and[DIG-dUTP]+[dTTP]must=[anyotherdNTP].Thereforetogeta1mMdNTPstockonemustdiluteDIG-dUTPstock1/10.
  • FIRSTdiluteseparate100mMdNTPstocksto10mM(eg.5ulto50ul,inwater).
  • THENmixequalvolumes(eg.10ul)of10mMdCTP,dGTPanddATPstock,and9/10thsvolumeofdTTP(9ul).Addequalvolume(eg.10ul)ofof1mMDIG-dUTP.
  • THENaddwaterto10vol(=100ul;add51ul):finalconcentrationeachdNTP=1mM;finalconcnDIG-dUTP=0.1mM,andofdTTP=0.9mM.

iii)USEmixmadeaboveat50uMeachdNTPinaPCRreactionmix,finalvolume25ul:

  • NEEDtodilutemix1/20;thereforeuse1.25uldNTPlabellingmixper25ulreactionvolume(1/20=5/100=1.25/25).

Tomakemastermix:multiplyamountofdNTPperreactionbynumberofreactions.


新闻动态
行业前沿
技术文章
最新产品