Fusion of two PCR products
Katie Ridd
Friday, 21 August 2009 09:00 UTC
Note from the moderators; this message has been re-posted to increase its visibility. It was originally posted in response to a previous forum question.
Does anyone have any suggestions for fusing two PCR products together that are different in size? One is 8kb and the other is 2 KB.
Everytime I try, I get 3 products, 10 kb, 8 kb and 2 kb. I would like to get more of the 10kb and less of the 8 and 2 kb.
Thanks so much
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Replies
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Hi Katie,
Assuming there is an overlap in the amplicons, you could try the technique in the following paper:
“Enzymatic assembly of DNA molecules up to several hundred kilobases”
http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html
Good luck!
John Gill
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Katie,
We have encountered this problem too when trying to stitch two PCR products together using a 20 bp overlap. In our case, because the two smaller template fragments had not been purified before running the second amplification, we presumed the unwanted amplifications to be due to residual primers being carried over into the second amplification reaction. The solution was simply to purify the desired target and reamplify. An alternative might be to purify the two smaller fragments before running the second amplification (if you haven’t already done this).
Sheldon
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