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Materials

  • 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.

Protocol

  1. 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.
  2. Take 1mL of overnight culture and inoculate 500mL LB broth.
  3. 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.
  4. 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.
  5. Gently resuspend the bacterial pellet in 1/4 volume of ice-cold MgCl2, taking 3-5 minutes for this procedure.
  6. Centrifuge the cell suspension at 4,000g at 4°C for ten minutes. Discard the supernatant.
  7. 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.
  8. Centrifuge the cell suspension at 4,000g at 4°C for 10 minutes. Discard the supernatant.
  9. Resuspend the cell pellet in 1/50 volume of ice-cold sterile 85mM CaCl2 in 15% glycerol w/v.
  10. 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 [1]:

  • 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
Error fetching PMID 12904643:
  1. Error fetching PMID 12904643: [Casali-2003]