Hi! I am having problems cloning a 1.6 kb fragment containing 2 different genes and 2X2A peptides into a vector that I already used to clone other fragments (digested with PmeI/BamHI). The vector contains 2A-GFP. I have sequenced the PCR product and it is correct, get lots of colonies after transformation but non have the insert. Have you encountered something similar?
Thanks for your answer, they are working, I used the exact same digestion of the vector to clone a different insert
You are obviously confident that your vector works fine. If it is the very same vector as before, your problem is the insert.
If it is a new digestion, Malik is right and you have to do a ligation without insert which should give no or few colonies.
If this is the case, you may just ligate some primer products, too small to detect by your restriction digest. Did you do a gel purification to avoid this?
In that case, I can only guess that your new plasmid recombines due to several identical 2A peptides. Then you may a E. coli strain that suppresses recombination events and grow your bacteria at a lower temperature.
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