- 1L Luria-Bertani (LB) broth
- XmL CaCl2
- XmL MgCl2
- Xml X% glycerol
We use E. coli 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. See below for a description of what these markers mean.
- Inoculate a 3mL overnight culture of E. coli cells (our genotype above) 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.
Competent-cell genotype description
Most of these descriptions are from :
- TetR = tetracycline-resistant
- Δ(mcrA)183 = Mutation in methylcytosine-specific restriction systems; allows more efficient cloning of DNA containing methylcytosine
- Δ(mcrCB) = Mutation in methylcytosine-specific restriction systems; allows more efficient cloning of DNA containing methylcytosine
- hsdSMR = mutations in Eco endonuclease activity, abolishing Eco restriction and methylation
- mrr = Mutation in methyladenosine-specific restriction system; allows more efficient cloning of DNA containing methyladenine
- endA1 = reduces activity of nonspecific endonuclease I, improving yield and quality of isolated plasmid DNA
- supE44 = prevents spread to natural E. coli populations; inserts Gln at amber (UAG) stop codons by supplying the tRNA glnV
- thi-1 = mutation in thiamine biosynthesis; requires thiamine for growth in minimal medium
- recA1 = homologous recombination abolished; prevents recombination of introduced DNA with host DNA, increasing stability of inserts
- gyrA96 = DNA gyrase mutation; confers resistance to nalidixic acid
- relA1 = eliminates stringent factor resulting in relaxed phenotype; allows RNA synthesis in the absence of protein synthesis
- lac = mutations in lactose metabolism; cannot utilize lactose as a carbon source
- Hte = unknown; enhances uptake of large plasmids
- F' proAB = mutations in proline biosynthesis; requires proline for growth in minimal medium
- F' lacIqZΔM15 = Allows α-complementation for blue/white selection of recombinant colonies in lacZ mutant hosts
- Tn10(TetR) = transposon insertion; encodes resistance to tetracycline
- Amy = amylase-producing
- CmR = chloramphenicol-resistant
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