PCR of Circular DNA
harald at mol.univie.ac.at
Wed Jul 30 22:07:18 EST 1997
In article <33DEACF2.44A at ns.east.cn.net>, ddsyr at NS.EAST.CN.NET says...
>Dear PCR Colleagues:
>Does anyone have the experiences about PCR the circular DNA like plasmid?
>that when using two primers in PCR reaction, could we get two products? I
>situation now when I PCR a plasmid pGEM3Z, I got two bands, one is 700bp,
>is 2 kb. I just wonder the reasons.
I am performing PCR regulary using a plasmid carrying my target sequence
as a positive control. I never ever got multiple bands with this, but if
you get them there are a few things you might check:
1) titrate the Mg2+ from 0.5 to 4 mM final concentration: moreMg2+ gives
2) use a higher annealing temperature
3) design more specific primers if possible
4) perform a Southern blot of your PCR product
5) perform a restriction digestion of your PCR product (say, cut into 1/3
and 2/3 length sounds reasonable, but of course your sequence determines
5) perform a nested PCR reaction with at least one (better two) nested
primers within the amplified sequence
6) do not despair
hope this helps and all the best for your experiments,
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