Karel Petrzik petrzik at entu.cas.cz
Thu Jul 4 02:35:31 EST 1996

Dear netters,

I have a problem with megaprimer in my PCR reaction. I have to use shorter PCR product (260bp) as an upstream primer and shorter 24bp downstream primer. I started with identical molar concentration of both primers and annealing temperature optimal for the shorter one. I have used touch-down PCR to annealing temperature 10 oC below the optimal Tan of the 24-mer. I need to prolong the 260 product to final 550 bp product. Here are my question: is necesarry to denaturate the dsDNA 260 bp product (megaprimer) before adding to the PCR reaction? Have any some idea how to solve this problem?

Thank all.
Karel Petrzik
Institute of Plant Molecular Biology
dept. Virology

petrzik at entu.cas.cz

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