I am inserting the orf of EMSY gene into lentiviral transfer vector pLVX (the EMSY orf was purchased from origene. pLVX was used to generate several constructs sucessfully in our lab). I have tried several methods, but always failed to get a correct clone of EMSY. At last, I got only one PCR-positive colony (this colony can grow but not well in LB broth), unfortunately, the EMSY orf contains three site-mutations. I have tried twice to make site-mutation to get the correct plasmid,but failed (no colony on plate). Then, I tranformed the pLVX vector containing wrong EMSY into DH5a and found no colony grow. It seems EMSY inhibits the replication of the vector.
It can happen - what if you cloned the EMSY sequence into a simple vector like pcDNA3? Does the same thing happen? Also, what competent cells are you using?
33 ng/ul for a midiprep is very low...it's a long shot but you could try using the ABLE series of competent cells. I did once get something to clone by varying the competent cell type, and I think it was ABLE K that finally did the trick.
Hey,
your construct seems to be quite big so the bgs need way longer to replicate the plasmid and therefore you should let them grow longer. Another things is decreasing the temperature to 30°C and to use recombinase deficient e.coli to avoid recombination events modifiing your constructs. For me it was normally only a matter of incubation time
regards
Sven
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning