Hello,

your PCR looks fine, although I personally never did the gradient PCR - usually, 70 degrees works fine for annealing of primers of such length. Also, doing 40 cycles might be an overkill - each cycles is, after all, an additional mutation risk. I usually do 30 cycles and thats enough. Additionally, high-fidelity polymerases seem to be ideal for this purpose.

An alteration to your protocol i propose is addition of a ligation step after the PCR. You can simply add ligase into your PCR mix, no need to change buffers. DpnI treatment comes after ligase inactivation.

Another useful alteration is the phosphorylation of primer's 5`-ends. This is done before the PCR to ensure the success of the subsequent ligation.

Hope this helps.

Thank you so much for the reply. I will surely try 30 cycles. For Dpn1 digestion i don't use the buffer; i just add the enzyme to the PCR mix (hope that's fine; forgot to mention about that). I will include ligation step after PCR (adding ligation buffer and ligase enzyme right?)

agree with @ Vladimir Shavva

regards

Thank you @Hassan Nima and @Vladimir Shavva

@ Keerthi Divakaran Yes, DpnI is PCR buffer works just fine. Ligase should be used in the same way - just add ligase enzyme into your PCR mix after the PCR, no need to add anything else.

@Vladimir Shavva Thank you so much for the clarification

@Vladimir Shavva But will the ligase enzyme work without the presence of ATP (which is supplemented in the ligation buffer) ?

@ Keerthi Divakaran Yes, you are right, you do need to add ATP (not the whole buffer though, as that will lead to salt concentration related issues) for ligation. My bad, i should have underlined that.

@Vladimir Shavva Thank you so much for the reply.

@Vladimir Shavva So i tried the alterations that you suggested. But nothing came out well. Now i am thinking of redesigning the primer (only one primer as its single primer SDM). The primer that i designed is 45 mer (unlike the 35 mer) and has Tm around 72 degree. I am quite curious to know whether the melting temperatures greater than annealing temperature affect the SDM or whether its fine. Please reply... thanks in advance.

@Keerthi Divakaran Unfurtunately this method is rather tedious and does sometimes require quite a bit of time and attempts.

I always set annealing tempreture at 70 for this kind of PCR.

May I inquire as to why you are only using one primer? To generate a functional plasmid you'll need to amplify both strands of it, so you need two primers, one for each strand. Have you tried to visualize PCR product using the agarose gel electrophoresis after the SDM PCR?