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PCR of entire plasmid followed by self-ligation for mutagenesis- what issues mig

plasmid pcr mutagenesis

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#1 ttt1

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Posted 21 April 2012 - 02:39 PM

I've used the quickchange method for quite some time and generally have decent results. Now I'm branching out and wondering, using standard primers with standard purification (ie, the cheap "regular" primers) oriented in opposite directions on a plasmid, followed by T4PK phosphoryation, DpnI treatment, and self ligation (appropriate purifications assumed)- will this method reliably lead to generation of plasmids with expected junction between the two ends of the pcr product?
Has anyone regularly used this method? I could imagine it would be nice for deletions of arbitrary size, insertions of short peptide tags, also even point mutant generation in cases where quickchange or other methods seem not to be working.
I had read that some people found plasmids that seemed to be derived from pcr products with "truncated" ends possibly due to mis-synthesized or incompletely synthesized primers.

#2 bob1

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Posted 22 April 2012 - 12:48 PM

The major problem is that PCR tends not to be very efficient for many plasmids due to the large(ish) size of the products. Otherwise, the scheme is fine, though it may be easier to just use regular cloning for inserting tags and the like.

#3 ttt1

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Posted 22 April 2012 - 05:51 PM

Well, I'd imagine it would be more efficient than the overlapping primer linear amplification method of quickchange. As in, you would generate at least as many molecules.





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