I transformed E.coli TOP10 cell with plasmid and left it to grow overnight including positive control. CAn it take more than 48 hours to see visual colonies or the transformation failedthe plasmid is compatible for successive transformation into yeast.
Hi Ruchi
In my experience, transformed E.coli Top10 (especially if commercial) do not take longer than 24h to form visible colonies, even when the plasmid is large or has a special sequence. If there are no colonies post 48h incubation (at 37 deg C), I would consider that as a transformation failure. However, did your positive control have colonies?
If not, that would be indicative of loss of transformation competency of the E.coli Top10 cells you are using.
Hope this helps.
Hi Ruchi,
as per aparajita said, E.Coli Top 10 do not take longer than 24 hours. If you keep incubate the plate longer than 48 hour you would not get the colonies or if you get some then there may be false colonies. As you have said there are no positive colonies observed then there must be problem with the Top 10 F cells competency, missing any step in the process. You can just check the concentration of plasmid, use fresh stock of antibiotic and also check the competent cell efficiency.
Have you got colonies yet from your re-do?
You mentioned earlier that you didn't even see colonies in POSITIVE-control plate neither. This indicated that either your competent cells were not working or your transformation procedures were not correct. Another possibility is that the 'antibiotic' in the plates was not correct, which kill them all.
E. Coli TOP10 does not take more than 16-20 hrs to colonies visualized after transformation. If any colony doesn't appear in the positive control plate then I think there is any problem in the transformation procedure or may be the TOP10 has loosened its competency. Need not to incubate more, because after 24 hrs of incubation false colonies will appear.
what is the optimum concentration of tetracyclin (from 10mg/ml stock) can be used to grow E.coli top 10 plain cells?
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