Maheshri:Competent

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.