Hello everyone, this is a question regarding signature tagged mutagenesis. I am trying to test the primers p2 and p4 ( primers to amplify the signature tags) from the initial plasmid pool containing tags (pUT mini Tn5Km2 plasmid with signature tags). But it doesn't amplify with PCR. On subsequent transformation and amplification of host DNA ( E.coli DNA extracted after successful mating with donor strain and selection on antibiotic plates) as template with same primers shows a beautiful 80bp band on the agarose gel. Any suggestions are appreciated. Thanks in advance.
Dear Sudharsan Gongati follow this basic article this may help you
Thank you Arvind. The paper is very informative and infact I have read it several times before. Still I cannot get around my head why the pcr doesn't work.
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