Problem with nested PCR

Reg

New member
Nov 16, 2015
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#1
Hi everyone. I have a problem with nested PCR. I keep getting my first PCR product in agarose gel that shows result of my second PCR. The band of first PCR product is faint when i did second PCR after gel purification. However, using the first PCR reaction directly for second PCR gave a strong band on the first PCR product. Although my second PCR product is fine,i want to get rid of the first PCR product. Any idea of how to do that?
 

Ivan

Administrator
Staff member
Nov 16, 2015
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#2
Hi Reg,

If I understand your situation you are getting the first PCR product re-amplified along with the second PCR product when you run your nested, second PCR, and you are trying to only get the second PCR product to amplify when you run the second PCR. Here are a couple of things you can do:



1. After you run the second PCR, run the PCR in an agarose gel and gel purify the PCR product you are interested. Assuming your two PCR products are different in size you would be able to separate them before cutting the one you want from the gel and purify it.



2. It is possible that you are getting re-amplification of your first PCR amplicon because you have some residual primers in your PCR reaction. After the first PCR either gel purify, or PCR purify (using a column), your PCR product before running the second PCR.



I hope this helps.



Ivan