Mutagenesis on PB2 part 2

[ericmjl]

@Starnite: Check out the report on the agilent reaction that has DMSO added to it. I think DMSO worked out.

After gel extraction, I replaced the A2 in our cloning kit box (-20ºC freezer), which only had 2 ng/µL of DNA.

My thoughts on the final missing part (PB2 part 1) are as such:

• It may be that the ~500 bp band from the latest +DMSO test is a non-specific annealing band that got favoured over the desired ~1600 bp band, and adding DMSO merely helped increase specificity for that band.
• I have a hunch that increasing back the annealing temperature to 68ºC might work, but if it doesn't, then we have a detour that may be worth trying.
• This detour is spiking Mn²⁺ ions into a MyTaq Red reaction. Manganese has been shown to increase the mutation rate of polymerases, and this might turn out to be a really cheap and convenient alternative to ordering the GeneMorph II kit - though, I will admit that I'm not sure if it'll work, which is why we get to try!
  • If it does, it'll turn out to be a nice and open source process engineering contribution: cheap, reliable mutagenesis for making sequence/protein libraries!

[ericmjl]

We decided in the end to just use MyTaq for the reaction. After all, since the last 800 bp of the PB2 protein are mutated, it's a good proof of principle.

Next week we can try doing the Mn²⁺ curve to see whether that affects mutation rate.