working on cloning of some viral genes into PfastBac1, vast amount of unexpected results directed me to insult the competent cells, so I had propagated it and performed miniprep, plasmid is found, I took the plasmid and transformed the cells again, it doesn't confer amp resistance. But unfortunately it gave the same PCR bands observed with some primers used in check cloning.
Is this normal?
the cell genotype is "F-φ80lacZ∆M15 ∆(lacZYA-argF)U169 recA1 endA1 hsdR17(rk-, mk+) phoA supE44 thi-1 gyrA96 relA1 tonA (confers resistance to phage T1)"
Of course no... It is mutant strain to be called competent... It must be a contaminated tube... I used to have this contamination by a yeast just similar colonies to the DH5... Especially if you work on it while it is still so small and young... Throw it away and get a clean strain... Otherwise, if you are sure about the tube you prepared the competent from... Try to use double destilled water... Usually water is a very nice source of a yeast contaminations... Good luck...
Are the bands familiar or just some non-specific bands? I agree with Ahmad that some contamination may be the reason for this. You can try to use a new set of competent cells and compare your results.
DH5 alpha T1 bacteria are not suitable for unstable plasmids. If you have huge inserts in the pFastBac, they are notoriously unstable, just because of the size of the resultant plasmid.
To confirm contamination of the competent bacteria, plate some on an ampicillin LB plate without any transformation and confirm if you have a lot of antibiotic resistant bacteria in your initial preparation of competent cells.
thanks to all replies,
i had tried another tube from the same package.
i had found no plasmid in.
and tested competent with 1*107 transformants/ug plasmid DNA.
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