Preparing chemically competent cells

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Making

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.
3. Let sit 30 minutes on ice.
4. Incubate cells for 30 seconds at 42^oC.
5. Add 1 mL SOC (Room Temp).
6. Incubate for 1 hour at 37^oC.
7. Plate 200 µL onto appropriate resistance plate.

TSS (50 mL)

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