3 replies to this topic

[kaveh]

Hi,

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?

many thanks,

K

[Papaver]

If you use complementary primers your product should be circular. If you are not quite sure than draw a model of the PCR with plasmid, direction of primers and you will see how your product will look like.

[shenzhou1015]

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.

[kaveh]

shenzhou1015, on 20 November 2011 - 08:20 AM, said:

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.

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.
thanks for the explanation.