ckafer at iastate.edu
Tue Jun 1 16:59:07 EST 1999
Here is the Inoue method in 6 easy steps.
Ultra competent E. coli preparation
(Inoue et al., Gene vol. 96 (1990) 23-28)
1. Grow an appropriate strain such as DH5 , XL1Blue etc. in 250 ml
SOB media at room temperature until ABS600=0.6. (This may take up to
2 days depending on strain)
2. Place flask on ice for 10 min. and then pellet the cells at 2500xg
for 10 min.
3. Resuspend in 80 ml ice cold TB and place on ice for 10 min.
Pellet the cells as before.
4. Resuspend the pellet in 20 ml ice cold TB. Add 1.5 ml DMSO.
Place on ice for 10 min.
5. Aliquot 100-200 ul into microfuge tubes and freeze in liquid N2.
6. Store the cells for up to 1 year at -70 C.
Use in a standard heatshock (42-45 deg. C) xformation.
SOB media=SOC media w/o glucose
TB (Transformation Buffer)
10 mM PIPES
55 mM MnCl2 ==> add this after pH adjustment
15 mM CaCl2
250 mM KCl
pH 6.7 with KOH
Filter sterilize or see note at end.
0.756 g PIPES
2.75 g MnCl2
0.551 g CaCl2
4.65 g KCl
200 ml H2O
pH 6.7 w/ KOH
H20 to 250 ml
Note: I have found that TB can be autoclave sterilized. However, the
MnCl2 should be dissolved in a small amount of water (don't forget to
subtract this amount from the other portion!), autoclaved separately
and then added to the rest of the TB when the solutions have cooled.
A dark brown precipitate will form during autoclaving if you don't do
On 24 May 1999 12:06:31 -0700, rmassol at CRIBA.EDU.AR (ramiro massol)
>I would like to get some protocols for making ultra-competent bacteria.
>I tried several
>times the standard chilled-CaCl2 and that doesn't seem to work very
>efficiently. I would really appreciate your help,
>National Biochemistry Research Institute
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