I am facing a MACH1 transformation related problem. My plasmid is PCMU-GAr which contains spectinomycin resistant gene. I've transformed the same plasmid in MACH1 and am getting the lawn of MACH1 after incubating the transformed cells on LB plates supplemented with 100ug/ml spectinomycin for 16 hr at 37 degrees. No plasmids were obtained later on. When I streaked MACH1 competent cell (untransformed) on LB plate supplemented with spectinomycin (100ug/ml), astonishingly the MACH1 cells showed growth. I am not sure about the fact if MACH1 is only able to do transformation when selection is ampicillin only. How to fix this problem?
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If untransformed comp cells are showing a growth on antibiotic plate, it means it is contaminated unless it has SpcR resistance gene in its genome.
Thanks, Chandru Subramani. The presence of SpcR in genome can be one possibility and definitely not eliminating the chance of contamination.
Later on, I used another vial of MACH1 competent cell (Untransformed) and this time from an old batch no growth was obtained when streaked on LB plates+ Spectinomycin (100ug/ml). I should have streaked old MACH1 on LB plates without antibiotics to check their viability because the older batch has gone through some serious temperature drop (nearly -6 degree) for 1 day. But being E. coli I feel the cells may lose their competence but are viable.
Will make a fresh batch.
It could be that MACH1 is SpecR or it could be that your spec plates are not good. I would make some fresh plates and test both MACH1 and some other E.coli control strain on the plates first.
Michael J. Benedik thank you for your suggestion. I will try using DH5alpha.
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