primers for SoE - (Aug/08/2005 )
I'm going to try creating recombinant genes using Splicing by overlap extension (this is to try to sort out the effect of mutations (and combinations of mutations) in 2 different proteins encoded by this gene. I cannot do anything else than putting the entire gene in an expression vector to create a virus that is capable of a single cycle of infection when cotransfected with a reportervirus that is deleted for my entire gene of interest).
When looking at the sequence, I have the choice of several primer- pairs (for the first PCR) , but I don't know exactly what combination exactly to choose.
I can choose between a combination of primers that on themselves do not have an overlap, but create about 100 bp overlap in the gene of interest.
Another option is to take primers that do have a small overlap, so if necessary I can make them both a bit longer so they have a bigger overlap.
What would be the best choice? Also, if the best option would be to make the longer primers, how long should the overlap be?
Thanks for every usefull hint.
I made a gene once (once!) by overlap extension, but not splicing overlap extension, so I don't know how relevent my tips are. (My gene was completely synthetic).
I used 12 overlapping 80mers for the construction, which overlapped by about 20-30bp. It was a pain in the a**, mainly cos my gene had alot of repetition so the damn oligo's kept going in the wrong place. My persistence (eventually) paid off though and I got a gene (completely non-functional - it didn't do what it was designed to do).
I guess what I'm trying to say is I'd do both, since you can't predict the outcome. Though if given the choice again, I'd outsource it to a company and make them construct the bloody thing! (BlueHeron in the US was one that I was considering before I left the project).
Bitter? Moi?? NEVER!