Freezing competent bugs...

L. Sumoy Sumoy at mbcg.uchc.edu
Fri May 13 10:34:24 EST 1994


>David L. Haviland Ph.D. wrote:

>...Normally, our lab has used the usual 50% (25% final) glycerol for freezing 
>bacteria.  Having recently found the Inoue et al Gene paper via Jim Graham, 
>I've been experimenting with freezing competent bugs (XL-1 mrf', and DH5-a) 
>in LB with 7% DMSO...
In our lab we have used with success the following procedure which uses
7% DMSO (and glycerol) in the freezing of competent cells.

Production of competent cells (from Hanahan, D. (1983). ÒStudies on
transformation of Escherichia coli with plasmidsÓ J. Mol. Biol. 166:
557-580):

2. Pick a single bacterial colony and disperse into 100 ml of SOB
medium.
SOB is:	for 100 ml
	2% Bacto tryptone	2 g
	0.5% yeast extract	0.5 g
	10 mM NaCl	200 µl of 5 M
	2.5 mM KCl	250 µl of 1 M 
		100 ml H2O
Autoclave.
Then add from a filter-sterilize stock:
	10 mM MgCl2	1 ml 1M 
	10 mM MgSO4	1 ml 1M
Incubate with vigorous shaking (275 rpm) at 37o C 2-6.5 hours until
OD550 is 0.45-0.55.
3. Spin in sterilized capped 25 ml Corex glass centrifuge tubes 10
minutes at 2500 rpm at 4o C in HB4 rotor.
4. Resuspend cells in 32 ml (1/3 volume) 1XFSB. Leave 5 minutes on ice.
1XFSB is:
	10 mM KAc
	100 mM KCl
	45 mM MnCl.4H2O
	10 mM CaCl2.2H2O
	3 mM HA CoCl3
	10 % redistilled glycerol
FSB (Frozen Storage Buffer) is prepared as a 5X solution without
glycerol; just before the procedure glycerol is added and buffer is
filter sterilized:
	50 mMKAc pH 7.0	5 ml of 1M (frozen stock)
	500 mM KCl	50 ml of 1M
	225 mM MnCl.4H2O	1x g
	50 mM CaCl2.2H2O	2x g
	15 mM HA CoCl3	3x g
		H2O to 100 ml
Adjust pH to 6.4 with 0.1 N HCl; should stabilize at 6.1-6.2. Store at
4o C.
To make 50 ml of 1X FSB:	10 ml of 5X FSB	
		5 ml of redistilled glycerol
		35 ml H2O
Filter sterilize.
5. Spin resuspended cells as above 10 minutes at 2500 rpm at 4o C.
6. Resuspend in 8 ml (1/12.5 original volume) FSB. Keep on ice.
7. Add 280 µl DMSO to 3.5 %; swirl and place on ice for 5 minutes.
8. Add additional 280 µl DMSO to a final 7%. Leave on ice 5 minutes.
9. Aliquot in 210 µl samples into screw cap 1.5 ml polypropylene tubes.
Highest efficiency is achieved if cells are used immediately.
For long term storage of aliquots, quickly freeze in dry ice. Store at
-70o C. Deep freezing preserves competence for high sub-cloning
transformation efficiency, which should be ³10+7 colonies/µg
supercoiled plasmid DNA good for routine subcloning.
Lauro.




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