PCR of Circular DNA

Harald Sigmund 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? 
I mean 
>that when using two primers in PCR reaction, could we get two products? I 
met this 
>situation now when I PCR a plasmid pGEM3Z, I got two bands, one is 700bp, 
another 
>is 2 kb. I just wonder the reasons.
>Ma
>


Hi, Ma!

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 
unspecific products

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 
this)

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,

Harald




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