TSS/KCM COMPETENT CELLS
Courtesy of Caleb Bashor
3-24-2011
TO PREPARE COMPETENT CELLS:
1. Grow a 5 mL overnight culture of cells in LB. The next day, outgrow by diluting 1:100 into 100 mL LB.
2. Perform outgrowth to OD600 0.4 - 0.5. Let culture sit on ice for 20 minutes.
Perform remaining steps at 4 °C.
3. Harvest cells by centrifugation. Remove supernatant and resuspend pellet in 4 mL of ice-cold TSS . Eliminate cell aggregates with thorough mixing. Let cell/TSS mixture sit on ice for 10 minutes.
4. Aliquot (100 µL) into pre-chilled eppendorf tubes and immediately snap-freeze in liquid nitrogen or ethanol/dry ice bath. Store aliquots at -80 °C.
TO TRANSFORM COMPETENT CELLS:
1. Thaw competent cell aliquots on ice. To 100 µL of thawed cells, add 100µL of ice-cold 1x KCM plus DNA (~200 µL total volume). If volume of DNA is greater than 5 µL, use 20µL 5x KCM + (x µL DNA) + (80 – x µL of ddH2O) for 200µL total volume.
2. Let mixture incubate on ice for 30 min. Heat shock at 42°C for 90 seconds (as a rule of thumb, Caleb does 45 seconds for every 100 uL of cells, which seems to work best for him). Plate cells or recover as appropriate.
For 100 mL 1x TSS (mix and filter sterilize---store at 4°C):
10g PEG 8000
3mL 1M MgCl2
5 mL DMSO
1x LB to 100 mL
5x KCM:
0.5M KCl
0.15M CaCl2
0.25M MgCl2