Difference between revisions of "Team:Bordeaux/Template:AchievementsProtocols"

Protocols

Making DMSO Competent Cells

- SOB : 1L

- Transformation Broth (TB) : 1L

1) Mix the Pipes, CaCl2, and KCl in 900 ml of millipore water.
2) Add NaOH until pH is 6.7 (Don’t worry, dust disappear after pH adjust)
3) Add MnCl2 (see above), stir, adjust volume to 1 L
4) Filter sterilize (Filters are in a cardboard box below the BET bench)
5) Store at 4C. 1L

DAY ONE :
1) Grow 12 ml overnight culture of favorite strain of E. coli in 2XTY (preheat medium at 37°C before inoculation)
2) Make SOB and TB.

DAY TWO :
1) Keep 5mL of SOB for initial OD.
2) Inoculate 1 L SOB with 12 ml overnight culture.
3) Grow culture at 18C (this temperature is really important as we see a 10-fold decrease in competency when we grow them at room temperature).

DAY THREE :
1) Grow cells until A600 0.5-0.7.

Subsequent steps should be carried out in the cold room on ice:
2) Put flask on ice for 10 minutes, then spin cells down at 2500xg (3350 RPM) in JLA 8.1 rotor
3) Pour off supernatant
4) Resuspend gently first in 25 ml TB, then add remaining 295 ml (Final 320mL)
5) Leave on ice for 5 minutes
6) Spin down again at 2500xg for 10 minutes
7) Resuspend cells in 40 ml TB
8) Add 3 ml of DMSO dropwise while gently shaking [final DMSO concentration is 7%].
9) Aliquot in 100 μl aliquots (you will need about 450 pre-chilled 0.5 ml tubes).
10) Flash freeze in liquid nitrogen.
11) Store at -80C (the lower the rack the better).