Dead E. coli problem

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nikinje
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Dead E. coli problem

Post by nikinje » Thu Feb 05, 2009 7:22 pm

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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Sepals
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Post by Sepals » Mon Feb 09, 2009 2:24 pm

Have you been keeping the bacteria at 37C the entire time? If so, it they have probably exhausted the nutrients. After 1-2 days of incubation you should place them at 4C.

Also E.coli, which naturally colonies the intestine rather than the body proper, prefers 36C to 37C (yes that little difference does matter).
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nikinje
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Re: Dead E. coli problem

Post by nikinje » Wed Feb 11, 2009 2:55 pm

I didn't know that about the 36 degrees, useful to know!

They were put in the fridge after an overnight growup (until I could just see the colonies). I think what has happened is that they have overgrown, used up the ampicillin on the plate and thrown the plasmid out so didn't re-grow on the new plate.

Another contributing factor is that I measured the temperature in the fridge today and it was 17 C .... The element was working so the sides felt cold but it wasn't making the centre of the fridge cold!

It is strange though as the DH5s didn't grow to the ridiculous sizes that the FusionBlue cells did, they were growing the rate you would normally expect even at 4 degrees. Maybe the FusionBlue are just better at growing at that lower temperature!

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