Our Tips to Help You Survive a Difficult PCR - Bitesize Bio

Secondary structures: secondary structures will keep your primers busy among themselves, instead of just annealing to your template. Luckily, ... Skiptocontent Iamsuremanyofyouhavebeenthere.Everythingisgoingsmoothly,andyourprojectseemstobeworkingoutperfectly. AndthenthereisthisonePCR. Forsomereason,itjustwon’twork.Itisablackdotonyourrecord.EventhoughIhaveascientificmind,Ihavetobehonest,Isometimesbelieveinwitches.Well,scientificwitches—bywhichImean:valid,solidreasons,forwhyaPCRisnotworking! ItissofrustratingtospendtimeandenergyonaPCR,andthengotocheckyouragarosegelfullofenthusiasmtofindnoband,alousysmear,ormanyunspecificbands. InthisarticleIwillgiveyousomeanswersthatmayhelpyoutosolveallyourproblems.Oratleast,yourPCRproblems. FirstThingsFirst…LookatYourTemplate Takeagood,hardlookatyourtemplate.Gettoknowasmuchaspossibleaboutit.DoesithavehighGCcontent(over65%)?Ifitdoes,beware.AhighGCcontentwillprobablymakeyourtemplatemuchhardertoamplify,butdon’tdespair,youcanaddressthis.Toimproveamplification,youmayincreasetheannealingtemperature,and/oraddDMSOoraddanothersecondarystructuredestabilizertoensurethatyourGCrichtemplatewillbeamplified. PrimerDesign Primerdesignisextremelyimportant.HerearejustsomeofthetipstokeepinmindwhiledesigningPCRprimers: Primerlength:Primersshouldbearound18–22nucleotidesinlength.Thisway,theyarelongenoughtoensurespecificity(bydecreasingtheprobabilityofbindingnon-targetsequences),yetshortenoughtobindtothetemplate. Secondarystructures:secondarystructureswillkeepyourprimersbusyamongthemselves,insteadofjustannealingtoyourtemplate.Luckily,therearesomebioinformatictoolsthatcanpredictthelikelihoodofsecondarystructureformationinprimers,suchasOligoAnalyzer3.1. Meltingandannealingtemperature:Meltingtemperature(Tm)iscalculatedusingtheAT:GCcontentofyourprimersandisprovidedtoyoubyyourprimersupplier.ForyourPCRtowork,thedoublestrandedprimersneedtobesinglestrandedinordertoannealtoyourtemplate—thisisaccomplishedbyheatingyourreactiontotheproperTm.PrimersthathaveTmsintherangeof52–58ºCgenerallyproducethebestresults.Annealingtemperatureisthetemperatureatwhichthesinglestrandedprimerswillannealtothetemplate:iftheannealingtemperatureistoohightheannealingprocesswon’tbesuccessfulleadingtolowPCRproduct.However,ifitistoolow,thennon-specifichybridizationmayoccur,whichleadstonon-specificPCRproducts. Specificity:Thisissuperimportant.Ifyourprimersarenotspecific,youcanneverbecertainoftheidentityofyourPCRproduct.Makesureyourprimerwillonlyalignwithyourtarget,andnotanyothersequence(e.g.,pseudogenes)inyourDNAtemplate!Youshouldalwaysblastyourprimersequenceagainstyourreferencegenomesequencebeforeorderinganynewprimerstominimizeproblemswithspecificity. Itisalsoagoodideatomakesureyourprimersdon’tannealtoanyknownpolymorphism/mutation.Rememberthatalleledropoutcanhappenifaprimerpaircan´tannealtooneallele,andthiscanleadtothelossofpreciousinformation.Itsoundsscary,buttherearemanybioinformatictoolsthatwillhelpyouaccomplishtheperfectprimers. TemplateConsiderations YoushouldtakeintoconsiderationthattheproblemswithyourPCRmighthavestartedlongbeforeyoueverstartedyourexperiment! YourDNAsample/templatemightcontainPCRinhibitors.Inhibitorscanbefoundinavarietyofbiological(e.g.,organs,blood)andenvironmentalsamplesorcanbecarriedoverduringnucleicacidextraction. Remember:TheDNAsamplemustbeasintactaspossibleifanyPCRistowork.Somedegradationistobeexpected,butaslongasyouhaveenoughintacttarget,youshouldbefine.Itisadvisabletocheckthequantity,purityanddegreeoffragmentationofyourDNAbeforeproceedingwithPCR.IfyourDNAistoofragmented,thenyouwilleitherneedtoredesignyourPCRtoamplifysmallerfragmentsorrevisityourDNAextractionprotocol. IfyouarehavingproblemswithamplificationandyoususpectthatthetemplateDNAistheculprit,thenIadviseyoutorunyourPCRinduplicate.Onereactionshouldcontainthecurrenttemplate,andtheothershouldcontainatemplatethathasworkedperfectlyinthepast.Remembernottoincludeanyotherdifferencesbetweenthesetworeactions! Whichbringsmeto… PositiveandNegativeControls Rememberthatincludingapositiveandanegativecontrolinyourexperimentisessentialfortroubleshooting,ifsomethinggoeswrong. Whathappenswhenyourpositivecontrolworked,butyoursampledidn’t?Well,maybetheDNAtemplatewasdegraded,orthealiquotwasn’tpreparedcorrectly.Ifthenegativecontrolshowsamplification,youmayhaveacontaminationofreagentsonyourhands! Nothingworkedatall?Sometimesyoujustneedtomoveawayfromthelabbenchandrethinkyoursteps,usingthetipspresentedhere.Don’tbeafraidtochangethingsuntilyoucanseethatperfectbandinyourgel.Butthinkcarefully,startslowly,andchangeonlyonefactoratatimeuntilyoufindtheculprit.Andbeforeyouknowit,youwillhavesurvivedthatdifficultPCR! TwitterFacebookLinkedIn WrittenbyCindyDuarteCastelão ImageCredit: BrettJordan 2Comments ThuyphamonApril5,2019at2:58am AnyonehasbeenusedLowMeltingAgarose?IhasbeenuseditforelectrophoresisinC.difficilePCRribotypingbuttheresultwasnotstable:loseband,smearbandandnotclearlysoIthinkagaroseandelectrophoresisisimportanttonotice LogintoReply EstebanBarilaonOctober12,2017at3:41pm DoesGCcontentmatterforcDNAtemplates?IfIamnotmistaken,cDNAissimplestranded,wouldyourecommendtouseDMSOstill?Ontheotherhand.HowdoIcalculateGCcontent?DoIusetheentirecdnamoleculeoronlythesegmentcoveredbymyprimers?IfIdothelatterIget70%GCcontent. LogintoReply LeaveaCommentCancelReplyYoumustbeloggedintopostacomment. ThissiteusesAkismettoreducespam.Learnhowyourcommentdataisprocessed. ScrollToTop