Does the E.coli DH5 alpha have an inherent ligase? If yes, is it possible that a restricted digested plasmid would get recircularized using the ligase from the E.coli DH5 alpha once its transformed and show up as a false positive?
I tried some ligations and as one of the controls, i transformed the cells with just the digested plasmid to check whether digestion was successful and found out very many colonies on the antibiotic seeded plate but the gel electrophoresis of the plasmid digest shows a single band as expected.So i was thinking of the possibility of internal ligase.
Dear Vikas Kukreja,
Of course there's all sorts of funny enzymes in your bacteria, including ligases which may lead to the results you've seen in your ligation/transformation. What makes me suspicious is the "very many" colonies you've observed, since I'd expect low frequency. What I would suggest is to do a dephosphorylation step on your vector before ligation. This will efficiently do away with your re-ligation issues. Protocolls can be found for any commercially available phosphatase enzymes. Also, if your digest is a double digest, I'd recommend controlling efficacy of each single digest separately.
Best regards,
Christoph
Hi Vikas Kukreja,
My answer is yes, because most of the DH5 alpha cells are easily ready for self ligated only vector but not insert. in this case the above suggestion will help you lot specifically for double digest.
Few things I may suggest you,
1. Check T4 DNA ligase enzyme as well as buffer activity, wait for the buffer completely thaw in ice bucket or 4C.
2. Another important aspect is the vector and plasmid ratio for ligation, we mostly used 3:1 if insert size is big, or even you can use 4:1 also.
3. The desire antibiotic plates must be not over 3 months before prepared. If long time storage plates most of the times give false positive colonies.
4. Finally, how long use incubate the ligation mixture into DH5alpha cells, i mostly suggest 45min is enough in 220rpm rotation in bacterial shaker. over growth may affect lot also again several false positive colonies.
This above suggestion will help you...
Good Luck...
All the above suggestions are something you should follow. I hope you are doing a minus ligase control (digested vector only and not phosphatase treated) and if then you see many colonies, it must be cellular ligases. I doubt it though. Good-luck
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