Team:SVCE CHENNAI/competent cell prep
Competent cells preparation
Requirements:
- Detergent-free, sterile glassware and plasticware
- Table-top OD600nm spectrophotometer
- SOB
- CCMB Buffer
Procedure:
- Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3. Use the "cell culture" function on the Nanodrop to determine OD value. OD value = 600nm Abs reading x 10
- Fill an ice bucket halfway with ice. Use the ice to pre-chill as many flat bottom centrifuge bottles as needed.
- Transfer the culture to the flat bottom centrifuge tubes. Weigh and balance the tubes using a scale
- Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.
- Decant supernatant into waste receptacle, bleach before pouring down the drain.
- Gently resuspend in 80 ml of ice cold CCMB80 buffer
- Incubate on ice for 20 minutes
- Centrifuge again at 3000G at 4°C. Decant supernatant into waste receptacle, and bleach before pouring down the drain.
- Resuspend cell pellet in 10 ml of ice cold CCMB80 buffer.
- Use Nanodrop to measure OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells
- Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
- Incubate on ice for 20 minutes. Prepare for aliquoting
- Aliquot into chilled 2ml microcentrifuge tubes or 50 μl into chilled microtiter plates
- Store at -80°C indefinitely.
- Test competence by measuring O.D
- Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles.
Sponsors
