Maheshri:Competent: Difference between revisions
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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. | 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. | ||
#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. | |||
#Grow at 37C until culture begin to look cloudy (~2hrs) | |||
#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). | |||
#Place cells in 4X50 ml conical tubes and put on ice for 10 min. | |||
#Spin 1.8Krpm (750-1000g) for 12 min in RC3B. Gentle harvest is important). Drain supernatant well. | |||
#Resuspend in 15 ml RF1 (cold) per 50 ml culture and incubate on ice for 45 min. | |||
#Spin as above | |||
#Resuspend in 3.6 ml RF2 (cold) per 50 ml culture and incubate 15 min on ice. | |||
#Place 100 µL of cells in precooled eppendorf tubes and freeze in liq. N<sub>2</sub>. | |||
#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 10<sup>6</sup>-10<sup>7</sup>, which is good enough for most cloning. | |||
==Solutions== | ==Solutions== | ||
| Line 33: | Line 24: | ||
RbCl 6 g 100mM | RbCl 6 g 100mM | ||
MnCl2-2H2O 4.95 g 50mM | MnCl2-2H2O 4.95 g 50mM | ||
KoAC 15 ml of 1M stock (PH7.5) 30mM | KoAC 15 ml of 1M stock (PH7.5) 30mM | ||
CaCl2-2H20 0.75 g 10mM | CaCl2-2H20 0.75 g 10mM | ||
Glycerol 75 g 15% w/v | Glycerol 75 g 15% w/v | ||
H2O up to 500 ml | H2O up to 500 ml | ||
| Line 50: | Line 36: | ||
MOPS 5 ml of 0.5M MOPS (PH6.8) 10mM | MOPS 5 ml of 0.5M MOPS (PH6.8) 10mM | ||
RbCl 0.3 g 10mM | RbCl 0.3 g 10mM | ||
CaCl2-2H2O 2.75 g 75mM | CaCl2-2H2O 2.75 g 75mM | ||
Glycerol 37.5 g 15% w/v | Glycerol 37.5 g 15% w/v | ||
H2O up to 250 ml | 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. | 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 (with date and name): | ||
==Modifications== | ==Modifications== | ||
6/10/05 Narendra Maheshri - Rather than freezing in LN2 cells, cells can be transferred immediately to –80C. | 6/10/05 Narendra Maheshri - Rather than freezing in LN2 cells, cells can be transferred immediately to –80C. | ||
Latest revision as of 11:54, 10 March 2011
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.
- 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.
- Grow at 37C until culture begin to look cloudy (~2hrs)
- 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).
- Place cells in 4X50 ml conical tubes and put on ice for 10 min.
- Spin 1.8Krpm (750-1000g) for 12 min in RC3B. Gentle harvest is important). Drain supernatant well.
- Resuspend in 15 ml RF1 (cold) per 50 ml culture and incubate on ice for 45 min.
- Spin as above
- Resuspend in 3.6 ml RF2 (cold) per 50 ml culture and incubate 15 min on ice.
- Place 100 µL of cells in precooled eppendorf tubes and freeze in liq. N2.
- 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.
