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Problems with Stratagene's site directed mutagenesis kit - Primers, PCR, Mutagenesis (Jun/16/2010 )

Hi all

I've been having problems with the Stratagene's site directed kit.. I've been trying to troubleshoot, and think its my primers 'coz I ran the control and it seems ok..WHY do primers for this kit have to be designed complementary to each other??? Previously whenever I'v designed primers they'v always been at two ends and never complementary.

So I'm wondering if primer dimers could be a problem. Currently my primers have a Tm of ~68 degrees. I do see primer dimer bands at the bottom of my gel and there is absolutely no amplification band. Tried transformation, nothing. I've repeated this about 4 times now..

Would really appreciate some info.

-4leafclover-

Quikchange is not an amplification reaction as such, it is more a way of introducing the mutation via the primers, so you don't end up with a bright band at the end of the cycling.

The primers are complementary as you are not trying to amplify a gene or short region of the plasmid, you are trying to replicate the whole plasmid. You are doing this by trying to anneal a primer (containing the mutation) to each strand after denaturation and then fill in the remaining sequence using the polymerase. this is followed by Dpn1 digestion that digests the methylated parent strand, leaving you with single-stranded (and linear) copies with the mutation inserted. If you want to improve the process efficiency, add a ligation step before the Dpn1 digest.

I don't bother running my reactions for SDM out on a gel any more, I just do the primer annealing/cycling step, ppt the DNA and clean it up, ligate and then digest with Dpn1 (in the same buffer, 18+ hours for both reactions), then transform. If you run some no primer reactions in parallel, these should give no colonies on the plates as a control for the primers working.

-bob1-

bob1 on Jun 17 2010, 05:33 PM said:

Quikchange is not an amplification reaction as such, it is more a way of introducing the mutation via the primers, so you don't end up with a bright band at the end of the cycling.

The primers are complementary as you are not trying to amplify a gene or short region of the plasmid, you are trying to replicate the whole plasmid. You are doing this by trying to anneal a primer (containing the mutation) to each strand after denaturation and then fill in the remaining sequence using the polymerase. this is followed by Dpn1 digestion that digests the methylated parent strand, leaving you with single-stranded (and linear) copies with the mutation inserted. If you want to improve the process efficiency, add a ligation step before the Dpn1 digest.

I don't bother running my reactions for SDM out on a gel any more, I just do the primer annealing/cycling step, ppt the DNA and clean it up, ligate and then digest with Dpn1 (in the same buffer, 18+ hours for both reactions), then transform. If you run some no primer reactions in parallel, these should give no colonies on the plates as a control for the primers working.


Thanks Bob

Q: Even if my primers were at two different ends, they would still amplify the whole plasmid because I'm allowing an extension time of 2min/kb. Wouldn't they?

I'm using a website: Northwestern university/ biotools that shows if your primers have any complementarity and unfortunately two of my forward primers happen to be complementary at a particular stretch. I'm wondering whether this would affect my reaction?

-4leafclover-

4leafclover on Jun 18 2010, 02:00 PM said:

Thanks Bob

Q: Even if my primers were at two different ends, they would still amplify the whole plasmid because I'm allowing an extension time of 2min/kb. Wouldn't they?

Depending on the size of the plasmid and the speed of the polymerase you are using (pfu I think for quikchange) then they should replicate the plasmid. You are actually trying to do this... think of it this way: You denature the plasmid into two separate single stranded circles of DNA, anneal a primer to each and then use the polymerase to copy the rest of the circle. Obviously, the polymerase can't ligate the end of the linear fragment it has generated to your primer, so you are left with a linear fragment and the parent circular DNA, which can then be used for another replication.

4leafclover on Jun 18 2010, 02:00 PM said:

I'm using a website: Northwestern university/ biotools that shows if your primers have any complementarity and unfortunately two of my forward primers happen to be complementary at a particular stretch. I'm wondering whether this would affect my reaction?

Possibly, self-complimentarity can be a problem. Try adding a secondary structure inhibitor such as DMSO to the reaction.

-bob1-