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E.coli culture in -4c for 2 hours means it will affect or not? what happened in

cloning molecular cloning

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#1 thil

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Posted 12 March 2014 - 12:34 AM

Hi,

I was wondering whether anyone had tried to use freezed E.coli culture (1ml - E.coli culture was stored at -40c for 2 hours)  prior to plasmid DNA inoculation (50 ml LB) and had success with the preparation? If so, what conditions did you use?

Thanks

S. Senthil Kumar



#2 bob1

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Posted 12 March 2014 - 11:44 AM

-4 is an unusual temperature to keep things at.  Refrigerators are usually +4 and freezers usually -20 (approximately). 

 

However, in general, unprotected bacteria will be damaged by having been frozen and thawed and may not be viable.  You can easily make glycerol stocks of bacteria using 15-20% glycerol that will withstand freezing at -80 and last for years.



#3 labtastic

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Posted 13 March 2014 - 05:33 PM

Situations like this are usually best answered by just giving it a go. See what happens and report back. We'll all learn something.  wink.png

 

I have seen undergrads freeze pelleted e. coli cells in a -20 with no cryo or buffer and they grew back up just fine.

 

The only times i get concerned about the quality of my cells is if I'm doing a transformation after some sort of cloning (mutagenesis, ligation, etc.) or expression (I usually like to use freshly transformed cells for expression rather than a frozen stock, though this isn't always critical).


Edited by labtastic, 13 March 2014 - 05:34 PM.


#4 bob1

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Posted 14 March 2014 - 10:34 PM

Note that while Labtastic is correct, it may work - it is not rigorous science to do this sort of thing - you may be unintentionally selecting for properties that you aren't aware of in your population of cells, such that they may change the results of your work (probably not for a plasmid extract, but you never know).

 

The general rule is - if in doubt, throw it out!  If you doubt your results from it before you use the plasmid, how will you know if the result you get later is a result of your problem earlier or the correct result!







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