ThisprocedureisusedtoanalyzeinsertsinpUC-derivedplasimids.

1.SUSPendE.colicoloniesharbouringplasmidsin50microlitersofTE.

2.Incubatefor5minat95degreesorinboilingwater.

3.Mixthefollowingsolutions.

Templatesolutionpreparedasabove10xTaqbufferdNTPmix(2.0-2.5mMeachof4dNTP)forwardprimerreverseprimerH2OTaqDNApolymerase

1ul1ul1ul2pmol2pmolupto10ul0.1unit

*Usually,allsolutionsexcepttemplatearepremixedinamicrofugetube.TheyaredispensedintothePCRtubescontaining1ulofthetemplatesolution.Forroutineuse,Imake1:1:7mixtureof10xbuffer,dNTPmix,andwatercontaining0.22(=2/9)pmol/uleachofprimerandstoreitat-20C.TaqDNApolymeraseisaddedtotherequiredamountsofthemixturejustbeforeuse,and9-microliteraliquotsaredispensedintoPCRtubescontaining1microliteroftemplatesolution.

4.PerformPCRfollowingstandardprocedure.TwentyfivecyclesareenoughforanalyzingpUC-derivedplasmids.

Solutions

TE

10mMTris-Cl(pH8.0)0.2mMEDTA

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