Taken from http://openwetware.org/wiki/TOP10_chemically_competent_cells

Preparing seed stocks

• Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C
• room temperature works well
• Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C
• room temperature works well
• 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
• This step may not be necessary
• Place in -80°C freezer indefinitely.

Preparing competent cells

• Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3
• This takes approximately 16 hours.
• Controlling the temperature makes this a more reproducible process, but is not essential.
• Room temperature will work. You can adjust this temperature somewhat to fit your schedule
• Aim for lower, not higher OD if you can't hit this mark
• Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.
• Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
• It is often easier to resuspend pellets by mixing before adding large amounts of buffer
• Gently resuspend in 80 ml of ice cold CCMB80 buffer
• sometimes this is less than completely gentle. It still works.
• Incubate on ice 20 minutes
• Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer.
• Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.
• Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
• Incubate on ice for 20 minutes
• Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates
• Store at -80°C indefinitely.
• Flash freezing does not appear to be necessary
• Test competence (see below)
• Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles.

Measurement of competence

• Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen)
• This is at 10 pg/μl or 10^-5 μg/μl
• This can be made by diluting 1 μl of NEB pUC19 plasmid (1 μg/μl, NEB part number N3401S) into 100 ml of TE
• Hold on ice 0.5 hours
• Heat shock 60 sec at 42C
• Add 250 μl SOC
• Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
• using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
• For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
• Ampicillin and kanamycin appear to do fine with 1 hour growth
• Plate 20 μl on AMP plates using sterile 3.5 mm glass beads
• Good cells should yield around 100 - 400 colonies
• Transformation efficiency is (dilution factor=15) x colony count x 10⁵/µgDNA
• We expect that the transformation efficiency should be between 5x10⁸ and 5x10⁹ cfu/µgDNA

Materials

• Detergent-free, sterile glassware and plasticware (see procedure)
• Table-top OD600nm spectrophotometer
• SOB
• SOC

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 DOWN to 6.4 with 0.1N HCl if necessary
• adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
• sterile filter and store at 4°C
• slight dark precipitate appears not to affect its function