FAQs

PCR

Troubleshooting
Ifnoamplificationproductsareobtained,whatparametersshouldbeconsideredfirstwhen
troubleshooting?
ForallTakaraClontechPCRPolymerases,considerthefollowing:
First,ensurethatallPCRcomponentswereincludedinthereactions.Apositivecontrolshouldalwaysbeincluded
toensurethateachcomponentispresentandfunctional.
Iftherewerenoproblemswiththeexperimentalsetup,increasethenumberofPCRcycles(35cyclesatatime)
upto40cycles.Increasingthecyclenumbercanovercomeissueswithlowabundancetemplateortemplate
inaccessibilityduetoimpuritiesinorpoorprimingefficiencyoftheprimers.
Ifincreasingthecyclenumberdoesimproveresults,thePCRconditionsmightbetoostringentfortheparticular
primersetortemplate.ConsidermodifyingthePCRconditionsasfollows:

Lowertheannealingtemperatureinincrementsof2degrees.
Increasetheextensiontime.
Increasethetemplateamount.Refertotheguidelinesprovidedwiththeenzymetodeterminetheoptimal
amountoftemplate.
ConsidertheseadditionalpossiblereasonsforPCRfailure:
PCRinhibitorsinthetemplatesample
IfPCRinhibitorsarepresent,usingdilutedtemplatemayincreasePCRefficiency.Alternatively,thetemplate
mayneedtobecleanedupusingakitsuchastheNucleoSpinGelandPCRCleanUpkit.Ifcleaningupthe
templateisnotapossibility,anenzymethathashighertolerancetoimpurities,suchasTerraPCRDirect
Polymerase,mayimproveresults.
Thetemplatehas>65%GCcontent
WhenamplifyingfromtemplateswithhighGCcontent,useanenzymeformulatedforthiscondition.Visitour
PCRselectionguidetofindanappropriateenzyme.
Primersarenotoptimal
Checkyourprimerscarefullyredesignifnecessary.Also,considerreamplifyingtheprimaryPCRproduct
using10folddilutions(1:100to1:10,000)usingnestedprimers.

WhenusingPrimeSTARHSDNAPolymerase,consider:
Usinganappropriateamountoftemplate.IfthetemplateishumangenomicDNAoracDNAlibrary,usenomore
than~100ngoftemplateina50lreactionmixture.
Usinganextensiontimeofatleast1min/kb.
Increasingtheconcentrationoftheprimers.

WhenusingPrimeSTARMaxDNAPolymerase,consider:
Adjustingtheextensiontimeifthereactionmixturecontainsexcesstemplate.Iftheamountoftemplateexceeds
200ngina50lreactionmixture,settheextensiontimebetween30sec/kband1min/kb.
Increasingtheconcentrationoftheprimers.

WhenusingSpeedSTARHSDNAPolymerase,consider:
 Increasingtheextensiontime.Althoughthestandardextensiontimeis10sec/kb,theextensiontimecanbe
increasedto~0.5min/kbforcomplextemplatessuchashumangenomicDNA.

Iftherearenonspecificamplificationbands,whatcanbedonetoimprovespecificity?
AllTakaraClontechPCRPolymerases

Issue:
Primersarenotspecific.

Solution:
UseBLASTalignmenttodetermineifthe3endsoftheprimersarecomplementarytositesotherthanthe
targetsite(s).RedesignprimersifnecessaryormodifyPCRconditions.

Issue:
PCRconditionsarenotsufficientlystringent.
Solutions:
Increasetheannealingtemperatureinincrementsof2degrees.
UsetouchdownPCR.
UseatwostepPCRprotocol.
ReducethenumberofPCRcycles.

Issue:
Toomuchtemplatewasused.
Solution:
Reducetheamountbyafactorof25fold.

PrimeSTARHSandPrimeSTARMaxDNAPolymerases

Issue:
Annealingtimeistoolong.
Solution:
Toachievespecificamplification,itisessentialtouseashortannealingtime(5to15sec)when
performingthreestepPCR.

PrimeSTARGXLDNAPolymerase

Issue:
PrimershavesuboptimalTmvalues.
Solution:
Toamplifytargets<1kb,designprimerswithTmvalues>55C,anduseanannealingtemperatureof
60C.IftheprimerTmvaluesare<55C,tryashorterextensiontimebetween5and10sec/kb.

TaKaRaExTaqandTaKaRaLATaqDNAPolymerases

Issue:
Nonspecificprimerannealingatlowtemperatures.

Solution:
Thehotstartversionsoftheseenzymesmayimproveresultsforsomeprimers.

SpeedSTARHSDNAPolymerase

Issue:
SmearingofthePCRproductbandsonagel.

Solution:
Excessivelylongextensiontimesmayresultinsmearing.Thegeneralrecommendationforextensiontimeforthis
enzymeis1020sec/kb.IfPCRyieldislow,tryincreasingthenumberofcyclesby5.
IfPCRgeneratesasmearafterrunningtheproductsonagel,whatcanbedonetoimprovethe
results?
First,determinethesourceofthesmearusingpositiveandnegative(notemplate)controls.Thiscan
determineifthecauseofthesmeariscontaminationorovercycling,orifthesmearresultsfrompoorly
designedprimersorsuboptimalPCRconditions.

Ifthenegativecontrolisblank,thereisnocontamination.Instead,thePCRconditionsneedtobe
optimizedconsiderthefollowingwhenadjustingthePCRconditions:

Reducetheamountoftemplate.
Increasetheannealingtemperature.
UsetouchdownPCR.
ReducethenumberofPCRcycles.
Redesigntheprimers.
Usenestedprimers.
Reamplifytheproduct.(Asmallplugofgelcanberemovedwithamicropipettetip,andtheDNAcanberecovered
byaddingtheplugto200lwaterthenincubatingat37C.5lofthissolutioncanbeusedforreamplification.)

Ifthenegativecontrolisalsosmeared,thereiscontamination.Youwillneedtodeterminethesourceof
contamination.ItmaybenecessarytoreplacePCRreagentsandtodecontaminatepipettesandyour
workstation(seequestionsbelowformoreinformationoncontamination).

WhataresomesourcesofPCRcontamination?
TherearefourmainsourcesofPCRcontamination:

1. ThemostcommonsourceofcontaminationisPCRproductsfrompreviousamplifications(calledcarryover
contamination).WhenlargeamountsofPCRproducts(1012 molecules)aregeneratedrepeatedlyoveraperiod
oftime,thepotentialforcontaminationbecomesincreasinglyhigh.
2. AnothersourceofcontaminationisclonedDNApreviouslyhandledinthelaboratory.
3. Sampletosamplecontaminationalsocanoccur.Thissourceofcontaminationismostlikelyinsamplesthat
requireextensiveprocessingpriortoamplification.
4. ReactionscanalsobecontaminatedwithexogenousDNAintheenvironment,includingDNApresenton
laboratoryequipmentandinreagentsusedforDNAextractionandPCR.

Howcancontaminationbeavoided?
ThesensitivityofPCRrequiresthatsamplesarenotcontaminatedwithanyexogenousDNAorany
previouslyamplifiedproductsfromthelaboratoryenvironment.Werecommendthatdistinctareasare
usedforsamplepreparation,PCRsetup,andpostPCRanalysis.

AlaminarflowcabinetequippedwithaUVlampisrecommendedforpreparingreactionmixtures.Two
stationsshouldbeestablishedthatarephysicallyseparatedfromeachother.

EstablishaprePCRareathatisforPCRreactionsetuponly.NoitemsfromthepostPCRareashouldbe
introducedintothisareathisincludesitemssuchasnotebooksandpens.
EstablishapostPCRareathatisusedforPCR,purifyingPCRamplifiedDNA,measuringDNAconcentration,
runningagarosegels,andanalyzingPCRproducts.

Equipmentshouldalsoberestrictedtotheseareas.ThePCRmachineandelectrophoresisapparatus
shouldbelocatedinthepostPCRarea.Havingpipettesandpipettetipswithaerosolfiltersdedicatedfor
DNAsampleandreactionmixturepreparationonlyisstronglyrecommended.Additionalrecommendations
include:

Havingseparatesetsofpipettesandpipettetips,labcoats,gloveboxes,andwastebasketsfortheprePCRand
postPCRareas.
LabelingpreandpostPCRitemssotheyarenotremovedfromtheirdesignatedworkarea.
FollowingthegoldenruleofPCR:NEVERbringanyreagents,equipment,orpipettesusedinapostPCRarea
backtotheprePCRarea.
PreparingandstoringreagentsforPCRseparatelyandusingthemsolelyfortheirdesignatedpurpose.Reagents
shouldbealiquotedinsmallportionsandstoredindesignatedareasdependingoniftheyareusedforprePCRor
postPCRapplications.ThealiquotsshouldbestoredseparatelyfromotherDNAsamples.

AcontrolreactionthatomitstemplateDNAshouldalwaysbeperformedtoconfirmtheabsenceof
contamination.Inaddition,thenumberofPCRcyclesshouldbekepttoaminimum,ashighlysensitive
assaysaremorepronetotheeffectsofcontamination.

HowcanIdecontaminateifIhavePCRcontamination?
1. LeavepipettesunderUVlightinthecellculturehoodovernight.UVirradiationpromotescrosslinkingof
thymidineresidues,damagingresidualDNA.
2. Sprayworkstations/equipment/pipetteswith10%bleachandthenwipeclean.
3. ChangetheworkstationsmovetheprePCRareatoanotherprecleanedlocation.
4. Donotuseanyinstrumentsorpipettesyouusedbefore.

WhatcanIdoifthePCRgenerateserrors?
ToavoiderrorsduringPCR,werecommendusingahighfidelityenzyme(seeselectionguide).In
addition,besuretoavoidthefollowing:

1. Overcycling
OvercyclingPCRreactionsoften:
ChangesthepHofthePCRreactioninamannerthatdestabilizesDNA.
IncreasestheamountofPCRproduct,therebyreducingtheefficiencyofthepolymeraseandpromotingerrors.
DecreasestheamountofdNTPs,therebyincreasingthelikelihoodofbasemisincorporationduetothe
unbalanceddNTPconcentration.(IfusingTakaraClontechPCRenzymes,thedNTPconcentrationis
optimizedto200nMincreasingdNTPconcentrationleadstomisincorporation.)
 CausesaccumulationofsinglestrandedanddoublestrandedDNA.
2. HighMg2+concentration
Mg2+concentrationrangesfrom15mM.UsingahighMg2+concentrationmayincreaseyield,butitalsomight
impacttheproofreadingactivityofenzymes.However,theMg2+concentrationshouldalwaysbehigherthanthe
dNTPconcentration.
3. TemplateDNAdamage
LimitUVexposuretimewhenanalyzingorexcisingPCRproductsfromgels.

http://www.clontech.com/US/Products/PCR/Resources/PCR_FAQs