random mutation during mutagenesis - will all colonies on LB plate have the same mutation? (Aug/26/2007 )

OK here is my question. I am using PCR to make a mutation of one amino acid in my gene. After I transformed, plated, grew culture, plasmid prepped and sequenced, I saw not only did I get my anticipated mutation. I also got a random mutation about 200aa away. Now I wonder will all of my other colonies on my Amp plate have the same random mutation, since they came from PCR, or can I pick another colony, grow it, sequence it and get a different result (hopefully without the random mutation) or should I scrap it and start over again? Naturally, the boss is in a super hurry for this construct.

I am quite suprised that I got the random mutation, I am using Pfu polymerase. Usually I get pretty lucky with my cloning and don't have random mutants, I guess it's my time to pay the piper?

Thanks for any help.

Assuming site directed mutagenesis was used.... well how many colonies did you look at? Was it just one or more then one.

If you have just looked at one colony, I would go look at another 3 more isolates (colonies) and see if I have the desired plasmid. Looking at one colony is hard to make a judgement. You could well be unlucky and picked a bad colone or there is a real problem... the second mutation migh actually be present in the template.

I did use site directed mutagenesis. I only looked at one colony, but usually thats all I need. I have checked the template and the second mutation is not the sequenced plasmid. I'll isolate a few more colonies and give it a go again, I was hoping to hear that maybe luck just wasn't on my side.

Thanks for the advice.

Hi,

I generally screen 5-10 colonies after site-directed mutagenesis.
Incidentally, make sure to use a polymerase with high fidelity (I use PFU Turbo). This will decrease the chance of a mutation being introduced randomly.

Good luck!