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By using a plasmid (interested insert) as a templete for PCR - PCR (Aug/05/2005 )

We have a couple of clones which have insert in each of the clones. The insert has been sequenced. We designed a pair of primers based on the sequence info, but there were no PCR products at all for some clones. My question is that if plasmid is used as templete, do we need to digest the plasmid first? Otherwise it will not be denatured during the PCR?



that is not necessary as the denaturation step for PCR will be sufficent for the PCR


You can also try amplifying with vector specific primers (eg T7/SP6) to check the insert. This doubles as a PCR control, to make sure you have adequate plasmid (and all other reagents) for amplification. I dilute my plasmid DNA 1:500 then use 1-2ul per 20ul PCR reaction. All other conditions remain the same (I use Ta of 55 C for T7 and SP6).

Don't forget that the T7 and SP6 primers produce a product WITHOUT an insert in most vectors, so make sure you take that into account if you are trying to check the size.

Hope that helps,