Team:Paris Bettencourt/Notebook/Drug Screening/Wednesday 3rd July.html

Drug Screening

ASDF

Wednesday 3^rd July

Chemically Competent Cells : BL21 (DE3) dCysI dFpr dydbk, NEBturbo stocks

Chemically Competent BL21 (DE3) Cells were made
We grew 5ml of sD001 cells overnight from a single colony. This 5ml was used to innoculate 500ml of LB broth.
Broth was incubated for 1.5h and optical density was read at 0.62 OD600.
Cells were alloquoted into 10 falcon tubes (50ml each) and centrifuged at 4000xg for 20 minutes at 4C.
Supernatant was removed and cells were resuspended in 200ml of Buffer 1 (4x 50ml) and left on ice for 20 minutes.
Cells were centrifuged again for 20 minutes at 4000xg.
Supernatant was removed and cells were resuspended in 12ml Buffer 2.
Cells were alloquoted at 500ul into microcentifuge tubes and frozen at -80C.
Buffer 1: 50mM CaCl2
Buffer 2:

• 0.53ml 2M CaCl2
  
• 2.8ml 60% Glycerol
  
• 8.67ml sterile H2O

This is the correct Chemical Competent Cells protocol (Similar to Protocol 2) , that we should have used instead of the one above:

1. Take a single colony of E. coli cells and inoculate 5ml of LB broth.
   Incubate over night at 37C and 200rpm and innoculate about 500ml to 1L sterile LB broth.
   DO NOT ADD antibiotics since these cells do not have a plasmid in them.
   Work as sterile as possible.
2. Inoculate about 300-400ml sterile LB broth with 500ul of preculture.
3. Grow the cells on a shaker at 37C until they reach an OD @ 600nm of 0.3 to 0.4 (1cm pathlength of the cuvette).
4. Centrifuge at 3000xg for 10 minutes at 4C. Ice down 100mM CaCl2 and 100mM MgCl2 solutions at this point.
5. Gently resuspend the bacteria pellet on ice in 50ml of ice cold 100mM MgCl2, taking 3-5 minutes for this procedure.
   Centrifuge the cell suspension at 3000xg for 10 minutes at 4C.
6. Resuspend the bacteria pellet on ice 50ml of ice cold 100mM CaCl2, incubate the cells for 10 minutes on ice.
7. Centrifuge the cell suspension at 3000xg for 10 minutes at 4C and resuspend the cell pellet in 4ml of ice cold, sterile 100mM CaCl2 in 15%(w/v) glycerol.
   Dispense in 50uL aliquots and freeze cells at -80C.

Chemically competent cells (NEB Turbo)
Prepared via iGEM chemically competent protocol with following changes.
500ul of preculture used to inoculate 500ml LB broth. Grown at 37C for 3h hours to an OD600 of 0.536.
Cells centrifuged at 3200 rpm on old centrifuge for 10m at 4C. Pellets resuspended in 80ml of CCMB80 buffer.
Incubated on ice for 20m. Centrifuged for 10m at 3200 rpm for 10m at 4C.
Pellets resuspended in 5ml of CCMB80 buffer. Aliquoted into Microcentrifuge tubes and frozen at -80C.