Competent Cells

Clemens Suter-Crazzolara, PhD un691cs at genius.embnet.dkfz-heidelberg.de
Thu Jun 4 02:21:32 EST 1998


On 3 Jun 1998 09:05:52 -0700, John Augliera said:

>Does anybody have a method for making bacterial cells competent and the pro
>per
>storage conditions.  Have tried a 10% glycerol method which has failed to
>work twice.

>Thank You


Will this help ?

cheers, clemens

11.1		Competent E. coli cells
Clemens Suter Crazzolara, 1996

efficiency: (HB101, DH5alfa) 1x 106 colonies / ug pBR 322

Remark: Cells stay competent at -70 for at least a year. The more tedious RuCl method did not 
proof to be much more efficient.

Materials:
Bake erlenmeyer at 200C (1-2h) to sterilize.
Rinse large centriguge tubes well with 70% alcohol to sterilize. Remove all alcohol, since trace 
amounts will kill the cells (either by rinsing with water or heating shortly).
 
LB-MM medium: All LB-medium is supplemented with 10mM MgSO4 and 10mM MgCl2 (add both 
Mg-solutions after autoclaving the medium)

For these solutions, use only purest water (e.g. Aqua Injectabili Braun, AIB):
50 mM CaCl2. Dissolve the appropriate amount of CaCl2 (culture grade) in AIB-water. Do not 
autoclave but prepare this solution under semi-sterile conditions. 
50 mM CaCl2, 20 % glycerol. Dissolve appropriate amount of  CaCl2 (culture grade) in AIB-water. 
Add 20 % glycerol (W/V). Do not autoclave but use a sterilization filter.
Store at 4C, cool on ice before use

Method
1. Single, fresh colony into 5 ml LB-MM, 37oC >275 rpm O/N
2. Add this culture to 500 ml LB-MM, use largest erlenmeyer available. 37oC >275 rpm 
3. Grow until OD600=0.3 (1 to 2 hours). I check this visually.
4. Keep on ice 10’ 15'. 
5. 10' 4000 rpm (from now on work in coldroom, keep cells <4 oC, proceed rapidly !)
6. Remove all supernatant, resuspend cells gently by pipetting in 1/2 volume of ice cold 50 mM 
CaCl2
7. Leave on ice for 1 h, spin 10' 4000 rpm in pre-cooled rotor.
9. The pellet smears along the wall: this indicates that the cells are getting competent. Pour 
off the supernatant, resuspend cells gently by swirling in 1/10th volume of ice cold 50 mM 
CaCl2, 20 % glycerol
10. split up in 0.5 ml aliquots, flash freeze in liquid N2 or dry ice + alcohol, store <  70 oC. 
11. transfect cells with 10 ng pUC19 and calculate competence as cells / µg pUC19




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