Drummond:Competent Cells

Materials

• 1L Luria-Bertani (LB) broth
• XmL CaCl₂
• XmL MgCl₂
• Xml X% glycerol

We use E. coli cells (ATCC: 55962) with the following genotype:

TetR, Δ(mcrA)183, Δ(mcrCB-hsd SMR-mrr)173, endA1, supE44, thi-1, recA1, gyrA96, relA1, lac Hte, [F' proAB lacIqZΔM15 Tn10(TetR) Amy CmR]a

• TetR = tetracycline resistant
• Δ(mcrA)183 = Mutation in methylcytosine-specific restriction systems; allows more efficient cloning of DNA containing methylcytosine
• Δ(mcrCB-hsd SMR-mrr)173 = = Mutation in methylcytosine-specific restriction systems; allows more efficient cloning of DNA containing methylcytosine
• 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
• lac 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

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

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.