Materials

• Detergent-free, sterile glassware and plasticware (see procedure)
• Table-top OD600nm spectrophotometer
• SOB
• CCMB80 buffer

Preparing glassware and media

• Eliminating detergent: Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic must be detergent free for these protocols. The easiest way to do this is to avoid washing glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware.
• Prechill plasticware and glassware: Prechill 250mL centrifuge tubes and screw cap tubes before use.

Preparing seed stocks

• Streak TOP10 cells on an SOB 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 microcentrifuge tubes. 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, but the temperature can be adjusted to fit 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 by swirling slowly over ice bucket. Be careful - rough resuspension could result in shearing that can dramatically lower cell competence.
• 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 microcentrifuge tubes. Store at -80°C indefinitely.

Note: 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 (Invitroge). 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 45 sec at 42°C
• Add 250 μl SOC
• Incubate at 37°C for 1 hour in 14mL overnight tubes in shaker to increase aeration
• Plate 20 μl on AMP plates and grow overnight. 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

Adapted from [http://openwetware.org/wiki/TOP10_chemically_competent_cells OpenWetWare]