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 Jessee patent as well.

Preparing seed stocks

• streak TOP10 cells on an SOB (no Mg) plate and grow for single colonies at 23 C
• Pick single colonies into 2 ml of SOB medium and shake overnight at 23 C
• Add glycerol to 15%
• Aliquot 1 ml samples to Nunc cryotubes
• Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes
• Place in -80 freezer indefinitely.

Preparing competent cells

• Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 23 C to an OD of 0.3
• Centrifuge at 3000g / 4C for 10 minutes in a flat bottom centrifuge bottle.
• Resuspend in 80 ml of ice cold CCMB80 buffer
• Incubate on ice 20 minutes
• Centrifuge again at 4C and resuspend in 20 ml of ice cold CCMB80 buffer.
• Incubate on ice for 20 minutes
• Aliquot to chilled screw top 2 ml vials
• Store at -80C indefinitely.

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

Experimental results:

• Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen)
• Hold on ice 1.5 hours
• Heat shock 45 sec
• Add 250 μl SOC
• Incubate at 37 C for 1 hour
• Plate 20 μl on AMP plates

Colony count was 420 on experimental plate. This is 420*15 = 6300 transforms/10 pg or 6.3 x 10⁸ transforms/μg. The control from Invitrogen was 210 colonies under identical conditions, or 3.2 x 10⁸ transforms/μg