Drummond:Competent Cells

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.
 * 5) *It may be easier to divide your cells into two 250mL batches, but it is not necessary.
 * 6) *While spinning, ice down 100mM CaCl2 and 100mM MgCl2 solutions.
 * 7) Gently resuspend the bacterial pellet in 1/4 volume of ice-cold MgCl2, taking 3-5 minutes for this procedure.
 * 8) Centrifuge the cell suspension at 4,000g at 4°C for ten minutes.  Discard the supernatant.
 * 9) 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.
 * 10) Centrifuge the cell suspension at 4,000g at 4°C for 10 minutes. Discard the supernatant.
 * 11) Resuspend the cell pellet in 1/50 volume of ice-cold sterile 85mM CaCl2 in 15% glycerol w/v.
 * 12) 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 Casali-2003 :
 * 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 &alpha;-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


 * 1) Casali-2003 pmid=12904643