I am trying to clone cDNA for a gene where the mRNA is 14kb. I have managed to PCR out the gene in 3 segments (5' 4kb-5Kb-5Kb 3') with unique restriction sites so that I can stitch the 3 segments together into an expression vector to get the full intact cDNA, two of the restriction sites are found naturally within the gene (AfeI and SexAI) and I introduced two myself, one at either end of the gene to allow me to put the cDNA into pCi. I TOPO cloned the segments and produced a multiple cloning cassette in pCi containing the restriction sites 5' KpnI, AfeI, SexAI and SalI 3' to allow me to stitch up the different fragments in the correct order.
I have managed to add on the 3' fragment using the SexAI and SalI restriction sites but I when I try to add the AfeI-SexAI segment all my colonies are false positives, I am using fastdigest restriction enzymes just so that I could use the same buffer for the different enzymes, I always leave the enzymes to digest for at least an hour, have also tried overnight but it doesn't help. I am transforming the digestion product using electroporation (using heat-shock gives no colonies which itself is weird as when I added the 3' fragment I used heat-shock to transform and got plenty of colonies and a high efficiency of positive colonies so surely the false positives should show up when using heat-shock??). I have also tried using CIP and get a reduction in colonies but they are all still false positives.
Does anyone have any ideas as to what could be going wrong or any different approaches I could try?
Edited by Mustafa, 30 July 2011 - 03:59 AM.