Hello,
I am a user of QuikChange II XL Site-Directed Mutagenesis Kit (Agilent) and I have performed SDM succesfully several times. I always introduce single nucleotide changes using a pair of mismatched complementary primers. According to the protocol, crucial part of SDM is to design primers with a melting temperature ≥ 78°C and of 25-45bp.
This time I have to deal with difficult template of very low GC content. Even when I pick very long primers (e.g. 55bp) for my DNA fragment, maximum Tm I can get for them is 73°C. I have checked Tm in several different tools available online and with regard to SDM reaction conditions. Primers reach Tm of 78°C with the length of about 100bp which is definately too long. I have also checked Tm in SnapGene that counts in a mismatch, it showed Tm of 64°C for 55bp primers.
Do you have any ideas how to optimize the SDM reaction so I could use primers with lower Tm (than it is said in the protocol)?
I was thinking of lowering annealing temperature but it might promote creating primer dimers (they are complementary and it happens in some level anyway) and I am afraid they would dissociate from the template during the extention step (68°C).
Thank you for any comments and ideas.
I would greatly appreciate your help!
Anna
If you get a pcr product then the primer must have been incorporated. Easiest is to run a temperature gradient and anything that amplifies to the right size should be roughly the right product and cloning will allow sequencing to ensure absolutely the right product. Primer dimer can be reduced by using a hot start enzyme and helped by using less primer. Primer dissociation is not a problem as the enzyme is active at all temperatures and most enzymes extend at more than 100 bases a second so even at the annealing temperature the primer will have extended from its original length by hundreds of bases even before you raise the temperature to the extension temperature
Hi, have you tried to use Agilent proprietary oligo design tool?
https://www.agilent.com/store/primerDesignProgram.jsp
Works very well to design primers for their kits!
Dear Anna,
having primers with Tm =73*C is ok. Running SDM PCR with an annealing temperature of 65*C or even 60*C should give you some of the desired product. In the end, you only need one colony.
Best of luck!
Hi Anna:
Re." Primers reach Tm of 78°C with the length of about 100bp which is definately too long", that's not necessarily so. I routinely use the QuikChange Lightning Kit, and for insertion of 24 - 40 bp tags, or inserting multiple mutations simultaneously, I have successfully used primers = 100-130 bp many times. Hope that helps,
Ron
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