Standard PCR reaction

• Lengthof18-24bases
• 40-60%G/Ccontent
• Startandendwith1-2G/Cpairs
• Meltingtemperature(Tm)of50-60^oC
• PrimerpairsshouldhaveaTmwithin5^oCofeachother
• Primerpairsshouldnothavecomplementaryregions

Tip:Primer3isanexcellentresourceforchoosingprimers.

Tip:Ifyouwillbeincludingarestrictionsiteatthe5""endofyourprimer,notethata3-6basepairspacershouldbeaddedinorderfortheenzymetocleaveefficiently.

1. Placethin-walledPCRtubesonice.
2. Fora50μLreaction,add:

   2μL	TemplateDNA(10ng-500ng)
   5μl	10XTaqbufferwithMgCl2
   1μl	dNTPmix(10mMeachnt)
   2.5μL	ForwardPrimer(10μMstock)
   2.5μL	ReversePrimer(10μMstock)
   0.2μL	TaqDNAPolymerase(5units/μL)
   32.8μL	Steriledeionizedwater(variable)

   Tip:IfyouaredoingmultiplePCRreactions,savetimebycreatinga"mastermix."

3. PCR:ThefollowingisatypicalPCRprogram.Theannealingtemperatureshouldbe5^oCbelowtheprimerTm.Theextensionstepshouldbe1-2minutesperkilobaseofproduct,dependingonwhetheryouareusingapolymerasewithproofreadingcapabilities.Seemanufacturer""sinstructions.

   Step1:InitialDenaturationfor2minutesat95^oCStep2:Denaturefor1minuteat95^oCStep3:Annealprimersfor30secondsat55^oC(or5^oCbelowTm)Step4:ExtendDNAfor2minutesat72^oCStep5:Repeatsteps2-4for25-30cyclesStep6:FinalExtensionfor10minutesat72^oC

4. Run2μLonageltochecksizeandconcentrationofPCRproduct.
   ReagentList:PCR

   Reagent	CatalogNumber
   TaqDNAPolymerase	NEB#M0267S
   Nucleotidemix(dNTPs)	NEB#N0447S
   Primers	www.idtdna.com

   Pleasenotethatthecatalognumbersgiveninthelistaboveareonlyexamples,andtherearemanyadditionalcompaniesthatsupplythesereagents.