Hello,
I am trying to made saturation mutations at two different positions. I used QuickChange II mutagenesis kit and it is very powerful (the single saturation mutagenesis works)
However, I added four primers into the reaction and run the PCR, the sequence result shows that either position one has been mutated or either position has been mutated. It was very disappointing that I could not find both two sites have been mutated.
I tried several ways to create double-site saturation mutagenesis library
However, neither of these works.
It should work in theory although I think you need very high efficiency for each mutagenesis step in order to get an adequate pool otherwise you will end up with very few of the double saturation mutants that you want.
If the two positions you wish to mutate are sufficiently distant, have you thought about making two pools of mutants and then using a restriction site in between the two sites to build a library combining the two pools of mutations.
Michael J. Benedik Thank you ,Michael! That's a good way to build the library. However, I found that there is no suitable restriction site for me to cut the plasmids....
When I grew up the colonies from the plate containing single-site saturation mutants for about 5 hrs and miniprep, I found the concentration is quite low. I was worried about if I grew them overnight, the bacteria may have contact inhibition.
You can introduce a silent restriction site with the same primer when you do one of the rounds of mutagenesis, or else just introduce into the gene itself by SDM before you create your first library pool.
5 hours of growth might not be sufficient, I don't think there would be any problem with growing a few additional hours until near saturation. However frequently I have just washed the colonies off the plate itself and mini prepped directly from the cells resuspended from the plate, that way there is no notable competition between clones during the outgrowth, beyond the difference represented by colony size on the plates.
In theory, I don't think neither the four-primers-at-a-time nor the tandem reactions strategy should work. I have not sat down with paper and pen to figure out all possible combinations but in the absence of strand displacement by the polymerase, they should generate non-overlapping ssDNA fragments containing one or the other mutation that can only be bridged by parental, dam-methylated DNA strands -and those hybrids should be digested away by DpnI.
The second strategy (one round of mutagenesis, transformation, purification of DNA en masse then a second round of mutagenesis) should work, but remember that the probability of finding the right clone will decrease dramatically if you don't enrich for recombinants; e.g. if both rounds of mutagenesis have an efficiency of 50% you'll find a double mutant only in 25% of the cases.
If the two sites you want to change are not too far away you can simply mutagenize both positions at once with one long primer pair. I've seen in the literature cases where this has been done with 90 b long oligonucleotides although have never done it myself.
Also, take into account that Agilent sells a different QuikChange kit for the exact thing you have in mind (simultaneous mutagenesis of several sites with separate primers). Despite having the same name it uses a different principle, and is called something like "multi-site quikchange"; just google it.
Hope it helps!
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