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Dead E.coli Problem

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#1 niki



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Posted 06 February 2009 - 03:35 AM

I've had a problem recently with the E. coli cells I've been using. I transformed 7 constructs into FusionBlue competent cells (from Clontech), they grew many colonies (significantly more than my control). I performed a colony PCR 5 days later and re-streaked the colonies, but not only did the colony PCR come up with nothing whatsoever (with 12 colonies taken from each plate), but the re-streaked colonies did not grow!

The plate I used for re-streaking was from the same batch as the ones used initially, so it wasn't a case of mixing up antibiotics. 5 days is not normally enough to kill E. coli cells.
This is only the 2nd time I have used these cells, so wondered if anyone else has any experience with them? (the time before worked perfectly with 8 constructs, positive colonies on the PCR and re-streaks growing fine).

They do seem to grow differently to DH5s. They grow very slowly initially in the 37*C incubator and produce tiny colonies, but when in the fridge these colonies overgrow very quickly; DH5s would grow to half the size in several weeks but these seem to grow very large within 2 or 3 days.

For the colony PCR I picked the largest colonies to avoid the possibility of satellites.

Anyone have any idea what happened to my E.coli? Did they just overgrow and die?

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