Cloning rare splice variants - Molecular Biology (Mar/04/2005 )
I am trying to clone full length rare gene splice variants and looking for any clues to improve my cloning. At the moment I am trying a simple approach of designing primers to the full length product, however obviously the predominant gene dominants the PCR reaction. I have thought about designing primers over exon boundaries and splice sites, however, I actually need the full length. Any suggestions would be appreciated.
I had similiar problems trying to clone splice varients. I tried running a PCR and cutting a band (not visible) out of the gel where the fragment should run then using this as the template in the next PCR reaction. Unfortunately this did not really work although I think the theory is still sound. It turned out to be easier to clone the transcript in a couple of parts-it really depends on your sequence and what convenient restriction sites there are.