Maheshri:Competent
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 supe. 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 200 µof cells in precooled eppendorf tubes and freeze in liq. N2.
10. Test competency: transform 100 µ 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):
6/10/05 Narendra Maheshri
Rather than freezing in LN2 cells, cells can be transferred immediately to –80C.
