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.
[http://openwetware.org/wiki/Preparing_chemically_competent_cells TSS cells]
Materials:
Preparation:
- 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.
- Grow the diluted culture to an OD600 of 0.2 - 0.5 (1-2 hours).
- 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.
- 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
- Centrifuge for 10 minutes at 3000 rpm and 4C.
- Pour / pipette off supernatant.
- Fully resuspend in chilled TSS buffer, using 10% of the culture volume that you spun down.
- Add 100 μl aliquots to your chilled eppendorfs and store at -80C.
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