Protocols for freezing E. coli

(Dave Johnston) daj at
Fri Jan 27 03:47:53 EST 1995


Our libraries are generating more clones than we can keep going, gridded 
out on plates so we want to freeze individual colonies from the primary 
plates to come back to later.

Ideally I would like to use 96 well, flat bottomed plates, place about 30 
ul of freezing mix (LB:glycerol @ 85:15) in each well, touch a colony into 
it, then grow @ 37C for a few hours to increase cell numbers (the primary 
plates are very dense and the colonies tiny) before freezing. Some questions

(1) Do E. coli grow in freezing mix? Yes, I could grow in LB and then add 
the freezing mix but this would use a hell of a lot of tips.

(2) Do you need to snap freeze E. coli or would placing the 96 well plate 
on a block of metal in the -70C freezer be good enough (large SA to well, 
minimal volume).

(3) Is polystyrene OK for -70C storage. Nalgene's data sheets suggest that 
it goes brittle below +20C! I could use thin wall polycarbonate plates (PCR 
machine types) but they are much more expensive and the well shape seems 
far from ideal.

Any ideas gratefully received. Thanks.

David A. Johnston
Research Fellow,
Dept of Zoology, The Natural History Museum, Cromwell Road,
South Kensington, London SW7 5DB. England
(tel 071 9389297, fax 071 9388754, email daj at

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