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Preparation of Competent Cells with DMSO

Get a printable version of this protocol (requires Adobe Acrobat) 

(Reference: Inoue, Hiroaki, Nojima, Hiroshi, and Okayama, Hiroto. "High Efficiency Transformation of Escherichia coli with Plasmids", Gene, 96, 23-28, 1990)

Streak frozen stock of cells on an LB agar plate:

BL21 DE3	LB
CJ236	LB
DH10B	LB
DH5alpha	LB
GM48	LB
JM109	LB
HB101	LB + Strr
N4830-1
NM522	LB
Solopak Gold	LB + Tetr
SURE	LB + Tetr
TOPP1,2,3,4,5,6
W3110

 

1. Allow the cells to grow overnight at 37°C
2. Pick 1 colony and inoculate 5 ml LB. Repeat so that you have a total of 3 inoculated cultures. Also prepare a blank LB control. Be sure to use sterile plastic pipettes during this step.
3. Allow the cultures to grow to saturation overnight.
4. Add 25 - 100 ul saturated culture to 250 ml SOB media, in a 2L flask. (Note: 2.5 ml of the magnesium stock solution filter sterilized through a 0.2 um filter, should be added to the SOB medium in the 2 L flask prior to this step)
5. Incubate the flask at room temperature with vigorous shaking.
6. Grow cells to A600 = 0.6; the specific growth rate, m, of SURE cells, for example, at R.T. ~ 0.44 h-1, and the double time at R.T. is about 1.6 h. It will take about 24 h to reach an O.D. of 0.6. However, this may not be the case the day you decide to make competent cells! It is best to constantly monitor their O.D. You can calculate the exact time the cells will reach an O.D. of 0.6 using the following formula: Time (h) = 1/m * ln (0.6/A600). The following chart demonstrates the approximate length of time for a culture to reach an O.D. of 0.6:    

BL21	22 hours
DH5alpha	30 - 37 hours
NM522	14 hours
SURE	17 - 23 hours

7. Place flask on ice for 10 min.
   
8. Transfer culture into a cooled centrifuge bottle and spin at 3500 rpm (~ 2100 x g) in the JA10 rotor for 10 min at 4°C.
   
9. Remove the supernatant and resuspend the pelleted cells in 80 ml of ice-cold buffer TB with a sterile plastic pipette; incubate on ice for 10 min.
10. Filter sterilze 100 ml of TB buffer. Store at -20°C.
11. Thaw 1.5 ml aliquot of DMSO. The DMSO must be high grade or the cells will not be competent. We order the molecular biology grade from Sigma and store it in 1.5 ml aliquots at -20°C.
    
12. Spin the cells down again in the same bottle at 3500 rpm for 10 min at 4°C.
    
13. Remove the supernatant and resuspend the pelleted cells in 20 ml of ice-cold buffer TB. Make sure the cells are well resuspended.
    
14. Add DMSO to the cell suspension to 7% (i.e. 1.4 ml).
    
15. Incubate the cells on ice for another 10 min.
    
16. Aliquot the cell suspension  (250 ul per eppendorf tube) and freeze the tubes quickly with liquid nitrogen.
    

This procedure gives cells with very high transformation efficiency - some claim as high as 10^9/ug of DNA.  We have certainly gotten 10^8/ug of DNA.  BUT - they don't store well...even at -80ºC.  We have never figured out why.

SOB Medium

(2% (w/v) Bacto Tryptone

0.5% (w/v) Yeast extract

10 mM NaCl

2.5 mM KCl

10 mM MgCl2

10 mM MgSO4)

10 g Yeast Extract 2.5 g NaCl 0.29 g KCl 0.09 g

	Dissolve in 500 ml of H2O
	Autoclave

Magnesium Stock Solution

1 M MgCl2	2 g
1 M MgSO4	2.5 g

	Dissolve in 10 ml H2O
	Filter sterilize
	Before use, add 1/100 vol of Magnesium stock solution, filter sterilized through 0.22uM filter, to the SOB medium (i.e. 2.5 ml to 250 ml medium). (FINAL=20mM).

Buffer TB:

10 mM Hepes

15 mM CaCl2

250 mM KCl

55 mM MnCl2

To make 100 ml: 

Hepes (M.W. 238.3) or PIPES	0.24 g 0.30 g
CaCl2 (M.W. 147.02)	0.22 g
KCl (M.W. 74.55)	1.86 g

	Dissolve in 80 ml of H2O
	Adjust pH to 6.7 with KOH
	Then add 1.09 g of MnCl2 (M.W. 197.9)
	Adjust the final volume to 100 ml with H2O
	Filter sterlize the solution