I have a problem with polycistronic-tRNA-gRNA cloning.
The relevant knowledge is in this paper.
https://www.pnas.org/content/112/11/3570
Inserts with multiple tRNA genes were integrated into the backbone and then transformed into MACH1. As a result of sequencing the plasmid obtained by mini-prep, there was only one tRNA gene.
I think this is because recombination took place in the com cell(MACH1).
The vector I make is larger in size than the paper above. So, when DH5a was used, the transformation efficiency was too low and failed.
What com cell can reduce recombination and have high transformation efficiency?
I think you're going to have to be more specific about your cloning process because I suspect the problem lies somewhere in there and not with what strains of E. coli you're using. Mach1 has a recA mutation so it shouldn't be any better at recombination than DH5a.
Also how large are we talking roughly? If you're using chemically competent cells and the plasmid is >20 kb I'd recommend switching to electro competent cells.
Alexandra Johnson
I made multiplex tRNA-gRNA fragments using golden gate assembly.
When gel electrophoresis was performed, this fragment was also the right size, and sequencing was also performed afterward.
The fragment and backbone were cut with restriction enzymes and cloned using T4 ligase.(Even after treatment with restriction enzyme, gel electrophoresis showed correct results)
However, the plasmid obtained here had only one tRNA.
This plasmid size is 14kb. I think Chemically competent cells have sufficient ability.
which part is the problem?
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