Freezer Storage of Cosmid Library in Microtiter Plates

(Dave Johnston) daj at nhm.ac.uk
Wed Jun 5 02:12:15 EST 1996


Michael, in my experience, bacteria will grow quite happily in 15% 
gllycerol (I am not sure they would like DMSO, it is certainly toxic to 
eukaryote cells, hence the need to wash them thoroughly when recovering 
from cryogenic storage in it). The things to watch out for are (1) that 
bacteria can use glycerol as a carbon source so if you let them grow up 
too much, they will start to use it, reducing the protective effect of 
the freezing mix you are growing up in (2) if you are using a standard 
incubator (ie not humidified), that the outer wells don't evaporate - we 
seal the plates individually in plastic bags to incubate them.

We routinely grow up for 4-5 hours in LB with 15% glycerol and then place 
plate on metal block in -70 freezer to fast freeze.

If you have lots of colonies, it might be worth considering 384 well 
plates, NUNC do some nice, generation ones with square wells of 
larger capacity than the original plates on the market with round wells 
(which were much more difficult to "hit" accurately with a pipette tip.



On 4 Jun 1996 16:14:46 
GMT,  Michael R. King writes:

>I am wanting to keep my unamplified cosmid library as individual clones
>rather than grouping them as a whole.  I want to put each clone in a
>microtiter well for long term freezer storage, that way I can use our 96
>well prong to put individual colonies on plates, hyb filters and other
>microtiter plates.  Does anyone know how to store them?  I would like to
>grow them up in the microtiter plates and then put them in the freezer.  At
>the moment I am growing some of the clones in a tube either in DMSO or
>Glycerol to simulate what I would like to do with the microtiter plate
>(Grow up directly in LB with glycerol or DMSO and put the plate directly
>into the freezer, that way I don't have to go back and add the
>glycerol/DMSO to each microtiter well after it has grown up in the LB).  Is
>any of this feasible? Or is there a better way?
>
>Michael R. King
>king1 at students.uiuc.edu

DAJ

David A. Johnston
Researcher, Biomedical Parasitology 
Dept of Zoology, The Natural History Museum, Cromwell Road,
South Kensington, London SW7 5BD. England
tel 0171 9389297 (from outside UK: 44 171 9389297)
fax 0171 9388754 (from outside UK: 44 171 9388754)
email daj at nhm.ac.uk



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