Ligation needed after SDM with PfuX?
Posted 04 November 2011 - 01:22 PM
I am trying to make a mutation in my plasmid (9.5 Kbp) using long complementary primers (PAGE purified) and PCR. I am using Invitrogen's PfuX. Now, is my PCR product going to be linear? If yes, do I have to do a ligation after PCR? Or it has to be after each amplification cycle?
I noticed there is no ligation step in Strategen manual, is that a different procedure?
Posted 20 November 2011 - 08:20 AM
- kaveh likes this
Posted 20 November 2011 - 08:35 AM
That's exactly what happened. I was surprised that I got a good success rate. I got some 3 mutated colonies in a screen of 12.
I have done such a similar SDM as you did. In fact, it's not needed to ligate the PCR products. After PCR, the DNA products are circular but with two nicks. The nicks will be ligated when they are transformed into E. coli.. But, Dpn I should be used before transformation. Or else, your SDM success rate will be very low.
thanks for the explanation.