I am cloning a transporter gene of about 4.5 kb long. PCR was successful, however, when I tried to clone it into traditianal cloning vectors, such as pUC series, the target cDNA always rearranged into about 3 kb. There are tandem domains in this gene. So I wonder this may be the problem. Now, what should I do? I already tried different E. coli strains, but the problem still exists.
Is there any other vectors suitable for this unique cDNA? Is pJAZZ will do? Or, are there any other cloning systems instead of E. coli, such as yeast? Please help me on this, thank you all!
may be the high copy number of pUC is a problem in your case. Try a low copy number!
Herve Levesque Thank you for your valulable advice. I think I will try it.
You mentioned that your sequence has tandem repeats. Apparently recombination could happen between these repeated sequences when propagating in E. coli. Have you ever sequenced the rearranged sequence(s) to see if this was the case? If so, use of a recombination deficient E. coli strain might solve the problem, but sounds like you have tried various E. coli strains already. I think the cloning vector(s) may be less of a problem, unless the vector(s) you used happened to contain sequences with some degree of homology to your sequence. Yes, transform into yeast may be a way. Good luck with your experiments.
Hua Xiao Thank you so much for your analysis. I didn't try recombination deficient E coli strains yet. Well, what if I want to construct an expression vector after gene cloning? It seems Gibson Recombinant Cloning will not work in this kind of E coli. Only Restriction endonuclease digestion and ligation works, right?
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