Drummond:Competent Cells: Difference between revisions
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==Materials== | ==Materials== | ||
* 1L Luria-Bertani (LB) broth | * 1L Luria-Bertani (LB) broth | ||
* XmL CaCl<sub>2</sub> | * XmL CaCl<sub>2</sub> | ||
* XmL MgCl<sub>2</sub> | * XmL MgCl<sub>2</sub> | ||
* Xml X% glycerol | * Xml X% glycerol | ||
We use cells with the following genotype: | |||
* TetR, Δ(mcrA)183, Δ(mcrCB-hsdSMR-mrr)173, endA1, supE44, thi-1, recA1, gyrA96, relA1, lac Hte, [ F', proAB, lacIqZΔM15 ,Tn10(TetR) Amy CmR]a | |||
Note that these cells are resistant to tetracycline and chloramphenicol, and are therefore not suitable for transformation with plasmids that carry TetR or CmR markers. | |||
==Protocol== | ==Protocol== |
Revision as of 19:48, 2 April 2009
Materials
- 1L Luria-Bertani (LB) broth
- XmL CaCl2
- XmL MgCl2
- Xml X% glycerol
We use cells with the following genotype:
- TetR, Δ(mcrA)183, Δ(mcrCB-hsdSMR-mrr)173, endA1, supE44, thi-1, recA1, gyrA96, relA1, lac Hte, [ F', proAB, lacIqZΔM15 ,Tn10(TetR) Amy CmR]a
Note that these cells are resistant to tetracycline and chloramphenicol, and are therefore not suitable for transformation with plasmids that carry TetR or CmR markers.
Protocol
- Inoculate a 3mL overnight culture of E. coli cells (our genotype below) in LB at 37°C. Do not add antibiotic. Work as sterile as possible.
- Take 1mL of overnight culture and inoculate 500mL LB broth.
- Grow this flask at 37°C for 3.5-4 hours until an OD600 of 0.3-0.4 is reached. Higher ODs will yield cells with impaired competence; lower ODs will result in fewer cells.
- Centrifuge these cells at 5,000g for 10 minutes at 4°C. Discard the supernatant; keep cells on ice.
- It may be easier to divide your cells into two 250mL batches, but it is not necessary.
- While spinning, ice down 100mM CaCl2 and 100mM MgCl2 solutions.
- Gently resuspend the bacterial pellet in 1/4 volume of ice-cold MgCl2, taking 3-5 minutes for this procedure.
- Centrifuge the cell suspension at 4,000g at 4°C for ten minutes. Discard the supernatant.
- Resuspend the bacterial pellet on ice in 1/20 volume of ice-cold CaCl2 and then add an additional 9/20 volume of ice cold CaCl2. Keep this suspension on ice for 20 minutes.
- Centrifuge the cell suspension at 4,000g at 4°C for 10 minutes. Discard the supernatant.
- Resuspend the cell pellet in 1/50 volume of ice-cold sterile 85mM CaCl2 in 15% glycerol w/v.
- Dispense in 100μL aliquots and freeze at -80°C.
500mL of starting culture yields X 100μL aliquots. One 100μL aliquot transformed with 1ng pUC19 routinely produces X colony-forming units.