Having sneaked around for a long time for answers for several of my lab-questions (thank you), I am now hoping for your help for my latest cloning problems.
I am currently trying to replace a puromycin gene in my vector backbone with GFP. The only unique RE-site near the puromycin gene is approx. 150bp in the gene. Therefore, I have not been able to use classical RE-cloning.
I therefore desided to use homolgous recombination (with approx. 16bp homology to the vector), hoping that it would not be a problem that there would be a long way from the site of homology to the end of the vector. The PCR amplified insert with vector-homology overhangs was succesfully created. However, recombination was unssuccessful despite several attempts and trying different conditions. I was using highly recombination effective E. coli.
I then tried to use my PCR amplified insert as a megaprimer based on the MEGAWHOP technique: http://barricklab.or...ew/Lab/MEGAWHOP
Please note, that my homology sequence is not as long as that suggested in the procedure. This did not work either, as I was not able to detect a PCR amplified product of the whole plasmid on an agarose gel. I tried doing the transformation any way, but had no colonies.
Not really knowing where to go from here, I have now a PCR running with my vector linearized in the puromycin gene and using my PCR amplified GFP with vector overhangs as primer, hoping to amplify a knicked vector. However, my hopes of this succeeding are very low.
Does anyone have any suggestions of where to go from here or anything I did not think about when designing my cloning setup(s)?
Kind regards, Emilie
Edited by EmilieKB, 02 July 2014 - 01:55 AM.