Team:Arizona State/Lab/Protocols/Competency

NEB cells

These cells can be ordered from [http://www.neb.com/nebecomm/products/category95.asp New England Biolabs] and should be stored at -80C. To transform, follow the NEB cell transformation protocol.

TSS^{[http://openwetware.org/wiki/Preparing_chemically_competent_cells]}

Materials:

• Cells (plated)
• TSS buffer
• LB broth

Preparation:

1. Grow a 5ml overnight liquid culture of cells in LB broth. After 12 hours, dilute this culture back into 25-50ml of fresh LB broth in a conical flask, diluting the overnight culture by at least 1/100.
2. Grow the diluted culture to an OD₆₀₀ of 0.2 - 0.5 (1-2 hours).
3. Chill X (where X is the volume in ml of your culture) eppendorf tubes so that they are cold when cells are aliquoted into them later. Chill TSS buffer.
4. Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.

All subsequent steps should be carried out at 4C and the cells should be kept on ice wherever possible

1. Centrifuge for 10 minutes at 3000 rpm and 4C.
2. Pour / pipette off supernatant.
3. Fully resuspend in chilled TSS buffer, using 10% of the culture volume that you spun down.
4. Add 100 μl aliquots to your chilled eppendorfs and store at -80C.