Maheshri:Competent

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Making Chemically Competent Bacteria

Protocol

From Arie Kaufmann

Note: This is a modified protocol that uses RbCl2 in addition to CaCl2. It takes some effort to make the two soln's (RF1 and RF2) but once made they last for a long time.

1. Pick 2-3 colonies from a freshly streaked plate, and inoculate into 2 ml LB (no Amp). it is important that the cells used are from freshly streaked plate and that they grow happily without any stress until harvested.
2. Grow at 37C until culture begin to look cloudy (~2hrs)
3. Inoculate the starter culture into 200 ml of LB and grow in a 3L flask at 37C until OD~0.4. Good aeration is important and cells should be harvested at an early log Phase (OD-0.4).
4. Place cells in 4X50 ml conical tubes and put on ice for 10 min.
5. Spin 1.8Krpm (750-1000g) for 12 min in RC3B. Gentle harvest is important). Drain supernatant well.
6. Resuspend in 15 ml RF1 (cold) per 50 ml culture and incubate on ice for 45 min.
7. Spin as above
8. Resuspend in 3.6 ml RF2 (cold) per 50 ml culture and incubate 15 min on ice.
9. Place 100 µL of cells in precooled eppendorf tubes and freeze in liq. N₂.
10. Test competency: transform 100 µL of cells with 1ng DNA and plate 1/1000, 1/100, or 1/20 of the transformed cells. Transformation efficiency is defined as the number of colonies per 1 µg DNA and should range between 10⁶-10⁷, which is good enough for most cloning.

Solutions

RF1

RbCl	        6 g	                        100mM
MnCl2-2H2O	4.95 g	                        50mM
KoAC	        15 ml of 1M stock (PH7.5)	30mM
CaCl2-2H20	0.75 g	                        10mM
Glycerol	75 g	                        15% w/v
H2O	        up to 500 ml	 

Adjust PH of RF1 to 5.8 with 0.2 M HoAC; filter sterilize using 0.22 µm filter and keep at 4C

RF2

MOPS	        5 ml of 0.5M MOPS (PH6.8)	10mM
RbCl	        0.3 g	                        10mM
CaCl2-2H2O	2.75 g	                        75mM
Glycerol	37.5 g	                        15% w/v
H2O	        up to 250 ml	 

Adjust pH of RF2 to 6.8 with NaOH if necessary, filter sterilize using 0.22 µm filter and keep at 4C. Modifications (with date and name):

Modifications

6/10/05 Narendra Maheshri - Rather than freezing in LN2 cells, cells can be transferred immediately to –80C.