I would like to substitute 3 aa (9 nt in total) with SDM using a single large primer to carry out multiple substitutions in a single run. I have designed the primers using the QuikChange Primer Design tool. They are 46 and 47 nt long (2 different templates) and ordered them salt free purified. The mutation of the 3 aa I would like to do are in the middle of the primer: 2 aa are next to each other then 3nt space between the second aa and the last one.
I have set up a first gradient PCR to check the annealing temperature from a range 50 to 70 degrees. My PCR conditions are as follows: 98 x 3 min, 98 x 20 sec, Tm x 12 mins (2 mins per kb...my plasmid is 6kb) for 40 cycles, 72 x 20 mins. I run the product on a gel and it doesn't work..
I have previously designed the primers using Agilent tool design (1 mutation substitution) and it worked straight away: 37 and 42 nt lond, salt free purified and same PCR condition (only difference 25 cycles instead of 40).
What is the problem? I hope you can help me.
You could try the Q5 site-directed mutagenesis kit from New England Biolabs. They also have a software to design the primers, which are not overlapping, but adjacent. The yield of the PCR is much higher. My advice is to column-purify (Qiagen or Macherey Nagel kit) the reaction product to get rid of the primers. Occasionally, I have got clones with the spurious integration of an additional primer between the ends of the amplicon.
I never use mutagenesis kit, they don't work better than just normal PCR conditions. I usually select around 20 b each side of the mutation and that's it. You could therefore try to design your own oligos, it might work better.
9 nt is also pushing it a bit, do you have to change all of these? Can you use the redundancy of the genetic code?
Also I am not sure why you need to use an annealing temperature this long (it does not depend on the length of the plasmid). I also use Phusion enzyme, not sure what you use.
In short to try: change of oligo, use phusion enzyme, minimise mismatch, reduce annealing time
If you are still out of luck with is you could try overlapping PCR, followed by InFusion cloning. This has worked 90% of the time for me.
Good luck
Hi,
what about using overlap extension PCR? I have done a ton of mutants with this kit of approach and it worked fairly easy and robust. We have changed up to 12 bases and added strecthes with up to 72 bases with this method
Sven
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