Adding 3'''' A overhang to a PCR product

Procedure

1. PurifythePCRproduct.BeforeaddingtheoverhangsitisveryimportanttoremovealltheProofreADIngDNAPolymerase(Pfu)bypurifyingthePCRproductcarefully(e.g.withacommercialPCRpurificationkitorphenolextractionandDNAprecipitation);sincetheproofreadingactivityofDNAPolymerasewilldegradetheAoverhangs,creatingbluntendsagain.
2. PrepareTaqDNApolymerasereactionmixforatypical20-50μlreaction:

   	FinalConcentration	Vol(μl)
   PurifiedPCRproduct	0.15to1.5pmol	Varies*
   dATP(10mM)	0.2mM	1
   PCRBufferwithMg(10x)	1x(1.5mMMgCl2)	5
   TaqDNAPolymerase(5U/μl)	1U	0.2
   ddH2O		to50μl

*TheA-additionreactionworksbestwhenaspecificamountofthePCRproductisused.Therecommendedamountis10–100ngPCRproductforeach100bplengthofthePCRproduct.Thiscorrespondsto0.15–1.5pmolPCRproduct(seetablebelow).

PCRproductsize	AmountofPCRproducttouse
100bp	10–100ng
250bp	25–250ng
1000bp	100–1000ng

3. Incubate20minat72°C.
4. ProceedtoTAcloning.Foroptimalligationefficiency,it""sbesttousefreshPCRproducts,since3´A-overhangswillgraduallybelostduringstorage.

Note

BasedonprotocolsfromQiagenandFinnzymes.