Blunting PCR products with Pfu.
hdang at cns.neusc.bcm.tmc.edu
Tue Jun 14 11:04:20 EST 1994
To people who are interested,
I do my PCR according to Barnes protocol, with a few variations depends on primers.
The PCR products contain considerable amount of "tailed" 3'-ends
which needs to be blunted before ligation to recircularize the mutant plasmid. After
checking on agarose gel of rxn that contain the desired product, add 2.5 units of Pfu
polymerase to the original 50-5=45 ul reaction and run the following temperature
80C, 1 sec > 72C, 15 mins
1-5 cycles, depends on the stability of product. Don't want to lose yield.
I was told that people at Stratagene also use a similar scheme for increasing their
blunt end cloning efficiency. They incubate at 72C for 30 mins. For the real careful
folks, purifying the products then add fresh buffer and Pfu may work even better.
Stratagene may have a 10X buffer for it too.
I saw an improvement of >5x #colonies compared to non blunted PCR-ligations, but I
have not done any optimization in terms of temp. and time. So don't be surprised if
it worked even better or worse.
Happy blunting! Hong
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