From 2007.igem.org

Make sure that the incubator (30/37C) and water bath (42C) are ON

Make sure required antibiotic plates are present. Check the antibiotic resistance on the plasmid map in Vector NTI.

Take the DNA out of -20 frig, let it thaw

Thaw the competent cells on ice for 7-8 min.

Add 1.0 µl of DNA (about 10ng) into the liquid (Don’t vortex). Tap the sides of the tube to mix.

Incubate the cells on ice for 30 min

Heat shock the cells for EXACTLY 30 sec at 42 C water bath.

Place on ice for 2 min.

Add 0.9ml of 37C S.O.C medium to each tube (S.O.C is made by dissolving 0.5 ml of 20% glucose in 25 ml of SOB. Make sure that the SOC is clear and not cloudy/ contaminated.)

Shake the tubes at 37 C, 280 rpm for 60 min or 30 C for 90 min

Incubate upright for 20 min., then upside down overnight (12-14 h) at 37 C or 16-18h at 30C.

Can leave the cells in the incubator for up to 18 hours but no more

Calculate the approx. S/N ratio of the transformation.

Decide on number of tries to be setup accordingly. Pick 12 tries if the S/N ratio is good (5:1), more otherwise.

Aliquot X ml (X = Y number of tries * 3.5, where Y = number of tries.) of TB into a clean (autoclaved) beaker or flask. Lb can be used if TB is not available.

Add X µl of appropriate antibiotic, i.e. a 1:1000 dilution to the above solution. The correct antibiotic should be added (same as the plate from which the colonies are picked.)

Take Y clean and autoclaved 14 ml polystyrene tubes (ones with thw white cap) and place them on a rack. Label them clearly. Aliquot 3.5 ml of TB/LB into each 14 ml tube.

Use a sterile wooden applicator (autoclaved) or pipette tip to carefully pick an individual colony and dip the colony end of the applicator / tip into a 14 ml tube. Repeat this Y times using a fresh applicator each time. Make sure to pick a single colony per try.

Place the tubes in the 37C shaker at 280-300 rpm and grow the cells for 12-14 hours. The tubes should be murky after the overnight growth.