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Inverse PCR product selection


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#1 labstud4

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Posted 14 March 2014 - 05:26 PM

Hello,

 

I am performing inverse PCR to delete portions of a plasmid (lets say the primers anneal with a 0,5kb gap). My process is as follows: inverse PCR ~25 cycles, DpnI digest, T4 PNK, T4 ligase, transformation, and finally colony screen. I was wondering whether anybody has tried modifying the protocol by adding DpnI after the initial 1 or 2 rounds of PCR in order to select for shortened primerF-primerR fragment. Theoretically, would this would reduce the amount full length linear product by a factor of 2?



#2 phage434

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Posted 14 March 2014 - 06:13 PM

I would not call this inverse PCR. It is just a normal PCR reaction followed by cutting the template with DpnI, phosphorylation of the product, and ligation, followed by transformation.

I have no idea why you think adding DpnI after the first few cycles will do much of anything special. The same fragments will amplify, assuming the first few cycles worked. You will have to use an enzyme leaving no A tails (nothing with Taq in it). The ligation will be a blunt ligation, and may cause issues.



#3 perneseblue

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Posted 16 March 2014 - 08:23 PM

I see no real point of adding DpnI after only 2 PCR cycles. I would just digest the PCR product after clean up with DpnI. Remember to do the PCR as with a proof reading polymerase (ie Phusion). AS phage434 cautioned do not use Taq or anything with Taq in it.

 

Blunt end ligation will only be an issue only your plasmid gets big ( >15kb). Using a diluted DNA solution for the ligation (compensated by a large ligation volume), will also encourage the DNA fragment to ligate to itself rather than to adjacent molecules.


May your PCR products be long, your protocols short and your boss on holiday




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