Preparing chemically competent cells

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Revision as of 11:33, 8 June 2005

Preparation

1. Grow a 5 - 25 mL culture to an OD₆₀₀ of 0.2 - 0.5.
2. Centrifuge for 10 minutes at 3000 rpm and 4^oC.
3. Remove supernatant and replace with 10% original volume TSS.
4. Make 100 uL aliquots and store at − 80^oC.

Using

1. Thaw TSS cells on ice.
2. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
3. Let sit for 30 minutes on ice.
4. Incubate cells for 30 seconds at 42^oC.
5. Add 1 mL SOC (2XYT is also suitable) at room temp.
6. Incubate for 1 hour at 37^oC.
7. Plate 200 µL onto plate with appropriate antibiotic.

TSS (50 mL)

• 5g PEG 8000
• 1.5 mL 1M MgCl2
• 2.5 mL DMSO
• LB to 50 mL