Cloning 2kb+ gene by PCR - Is it doable? (Dec/02/2007 )
I am trying to clone a certain gene (translated region from ATG to UGA is over 2000 bp) by PCRing it from a cDNA library. Can I do it in one piece, or should I PCR it in two pieces and assemble them afterwards? Or maybe I shouldn't bother and get EST clones for cloning? Any tips and suggestions appreciated.
in theory this should be easy, but of course it depends on some factors such as your primer design, expression level ...
but I have cloned fragments up to 5 kb made by PCR, so 2 kb should be feasible
just don't forget to use a proofreading enzyme
Yeah, that should be fine. If you're not sure whether you can get it one hit order the primers for amplifying it in two pieces as well as the outside ones and try both. You'll need internal primers for sequencing anyway with a fragment of that size. Try cDNA first. Use a couple of tissues that show high expression of the gene. cDNA is generally cheaper than a clone but sometimes clones are cheap so check out the prizes. Ditto on a proof-reading polymerase, Phusion is the best.
PCR for 2kb product should work but may need a little optimization. Also check for EST clones. If you do find one, then may b its the easier route.