TOP10 chemically competent cells

This protocol is a variant of the Hanahan protocol using CCMB80 buffer for DH10B and TOP10 strains. It builds on the Jesse patent as well.

• streak TOP10 cells on an SOB (no Mg) plate and grow for single colonies at 23 C (patent says 30C)
• Pick single colonies into 2 ml of 15/10 medium and shake overnight at 23 (30) C (Hanahan uses SOB no Mg)
• Innoculate 200x volume of 15/10 medium with the overnight culture and grow to an OD of 0.3 (Hanahan uses SOB no Mg)
• Centrifuge at 4C and resuspend in 1/3 initial volume of ice cold CCMB80 buffer (skipped in patent)
• Incubate on ice 20 minutes (skipped in patent)
• Centrifuge at 4C and resuspend in 1/12 of the initial volume of ice cold CCMB80 buffer.
• Incubate on ice for 20 minutes
• Aliquot to chilled Nunc cryotubes, and freeze in dry ice/ethanol by placing the cryotubes in a ziplock bag and immersing the bag for at least 5 minutes
• Store at -80C indefinitely.
• Note: There is disagreement on temperature. Patent says 30C, Hanahan says lower temperatures may help, ref to Inoue)

CCMB80 buffer:

• 10 mM KOAc pH 7.0 (10 ml of a 1M stock/l)
• 80 mM CaCl₂.2H₂O (11.8 g/l)
• 20 mM MnCl₂.4H₂O (4.0 g/l)
• 10 mM MgCl₂.6H₂O (2.0 g/l)
• 10% glycerol (100 ml/l)
• adjust pH to 6.4 with 0.1N HCl

15/10 medium:

• 1.5% yeast extract
• 1% Bacto-Tryptone, 10mM NaCl
• 2mM KCl
• 10 mM MgCl₂
• 10 mM MgSO₄
• Note: There is disagreement on the Mg concentration of this buffer Patent says Mg; Hanahan says no Mg.