Mutagenesis woes - (Apr/27/2010 )

I did something very silly. I setup my mutagenesis reaction, pipetted reagents, and loaded pcr machine. I usually let tubes sit at 95degC for 5 mins before starting the cycles....well I forgot to do that and my sample sat at 95degC for 2 hours. When I remembered I quickly jumped in my car to race back to the lab and hit the resume run button. I let it complete cycling, set aside 5ul, and digested with DpnI. I ran undigested against digested, both have a band (yay!).

My question is this: did I trash my DNA or do you think my mutagenesis actually worked like the gel suggests and go ahead with the transformation?

It is interesting that despite the 2 hour incubation at 95oC your reaction worked since after such a long incubation most of your Taq would be dead. The fact that you ran a digest and got the results you expected suggests to me that your reaction worked (there was enough Taq alive and your reaction was robust enough) so you should go ahead with your transformation.

Good luck.

I am using Pfu Ultra polymerase, not Taq. I thought that the heat would kill the enzyme too, it's resilient apparently. I was more worried about the DNA being destroyed.

Interesting...

I would say go ahead and do the transformation. If the transformed cells grow, pick a single colony and do a miniprep, sequence the plasmid. The cost is relatively small and the result can be really convincing.

Why not program the thermal cycler to automatically go into the cycling after the 5-min 95C incubation?