Preparing competent cells (XL-1 Blue or M15[pREP4]) (MSKC)
Adapted from Quiagen-The Quiaexpressionist

1. Using a toothpick, streak out a plate of cells on appropriate LB/drug plate
(100 mg/mL Amp for XL-1 Blue or 100 mg/mL Amp & 25 mg/mL Kan for M15)
2. Pick a single colony of cells and inoculate 10 mL of LB-drug broth
3. Grow shaking overnight at 37°C

Adhere to the following steps exactly!!!

4. Add 1 mL of culture to 100 mL of prewarmed LB /drug broth in a 250 mL flask
5. Shake and grow at 37°C until an OD of 0.5 is reached
(~4 hours for M15, ~3 hours for XL-1 Blue)
6. Cool culture on ice for 5 minutes, transfer culture to round bottom centrifuge tube
7. Collect cells by centrifugation (5 min, 4000 x g, 4°C)
8. Discard the supernatant. Keep cells on ice.
9. Resuspend the cells in 30 mL cold TFB1 buffer and incubate on ice for 90 minutes.
TFB1-100 mM RbCl, 50 mM MnCl₂, 30 mM Potassium acetate, 10 mM CaCl₂, 15% glycerol, pH 5.8, filter sterilize
10. Collect the cells by centrifugation (5 min, 4000 x g, 4°C)
11. Discard the supernatant. Keep cells on ice.
12. Resuspend cells in 4 mL of ice cold TFB2 buffer
TFB2-10 mM MOPS, 10 mM RbCl, 75 mM CaCl₂, 15% glycerol, adjust to pH 6.8 with KOH, filter sterilize
13. Prepare 100-200 µL aliquots in microfuge tubes and store at -80°C immediately