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mutagenic primers with very high GC content.


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#1 BHABA

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Posted 25 June 2013 - 06:11 AM

Hello guys,

I am trying to introduce a few point mutations, using an inverse PCR method (analogous to Stratagene QuikChange Sitedirected Mutagenesis). I have got my mutagenic primers synthesized from Macrogen Inc, and carried out the reaction manually (did not use any kit). I did manage to get a few positive clones, however a couple of mutants just would not come.

Interestingly for these two cases, the primers are of very high GC content (>70%). I have tried with 1% DMSO in the reaction mix, however, it still fails.
Moreover, when i did see colonies, they turn out to be false positives. I am using NEB DpnI for digesting the parent plasmid post PCR amplification, and it seems to be working fine.

Can anyone suggest where the problem lies?

I will be happy to provide any other information regarding the reaction mix if needed.

Note: I use desalted primers, and carry out PCR cleanup prior to DpnI digestion

regards


bhaba kd

#2 phage434

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Posted 25 June 2013 - 11:24 AM

You could try 2-5% of a 1M betaine solution, which in my experience works wonders for high GC reactions. Be sure to use a non-strand displacing enzyme.

#3 BHABA

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Posted 30 June 2013 - 10:23 PM

Thanks a lot. I will try out the suggested modifications. I have been using Phusion Polymerase for my reactions, and notable I did manage to get a few mutants done. Can I still continue with it or do I need to find an alternative?

#4 phage434

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Excellent

Posted 01 July 2013 - 06:28 AM

Phusion should be fine.




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