Hi, I have been trying to clone topoisomerase-3B from C elegans cDNA in NEB 5-alpha Competent E. coli. I performed hifi assembly of the top-3B cDNA insert and RE digested yeast two-hybrid pGBD vector and transformed it in e. coli. However, none of my transformed colonies on the selection plates have the plasmid with top-3Binsert. Does anyone have any suggestions for cloning and transforming top-3B in E. coli?
What controls did you do and what did they tell you? Perhaps your recipient vector wasn't completely digested, or perhaps your ligation failed, or perhaps the assembly failed. It is impossible to diagnose without knowing details.
I know it's expensive, but use one of the TOPO cloning enzymes. Just add your PCR product, no need to do a digest first. It's worked every time for me, even for really big insert (~8kb).
Good luck!
Thank you Katie A S Burnette for the suggestion. With the TOP O cloning method, how common are the vector-only(no insert of interest) colonies on the selection plate? My main worry is that since I am trying to clone an essential gene top-3B which might be tightly regulated in the E coli itself, will trying to clone the topoisomerase enzyme in E coli increase the DNA damage or toxicity in the E. coli such that no e coli containing the plasmid with insert will survive? Is there a way to troubleshoot the toxicity aspect of cloning TOP-3B? Othman Mueen Mohammed Michael J. Benedik, in the same RE digested vector, with the same Hifi mix kit and protocol I was able to clone a VBH-1 cDNA insert, transform in competent cells, and get positive transformants containing my insert. Still, only TOP-3B cloning has been unsuccessful despite multiple attempts.
TOPO vectors are just for cloning, they are not expression vectors. You move into a different vector to induce protein expression.
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