NKI standard Method

This protocol is for cloning strains eg. NovaBlue or DH5a. There is no need to drop the temperature for expression strains such as BL21 (DE3) and this will not give you more competent cells. These cells can be made by the same protocol but the temperature can rem remain at 30°C or 37°C until OD600 is exactly 0.6.

Materials required

SOB medium: Per litre:

• 20 g tryptone
• 5 g yeast extract
• 0.5 g NaCl
• after autoclaving the above
• 2.5mM KCl
• 10mM MgCl2
• 10mM MgSO4
• pH6.7-7

TB buffer (50ml)

• 10mM Pipes ~ 0.15g
• 15mM CaCl2 ~ 0.11g
• 250mM KCl ~ 0.93g
• adjust pH to 6.7 with NaOH
• 55mM MnCl2 ~ 0.55g

Procedure

1) Inoculate from an overnight grown in LB: 1ml cells diluted in 100ml SOB + appropriate antibiotics.

2) Grow cells at 37°C until 0.05 < OD600 < 0.3 Then drop temperature to 18°C for growth until OD600 is exactly 0.6

3) On ice for 10 minutes.

4) Spin at 3000rpm (1000g) for 10 minutes at 4°C.

5) Resuspend cells gently in 33ml of ice cold TB.

6) On ice for 10 minutes.

7) Spin at 3000rpm (1000g) for 10 minutes at 4°C.

8) Resuspend cells gently in 8ml of ice cold TB.

9) Add DMSO to a final concentration of 7% à 0.56ml DMSO

10) Place on ice for 10 minutes

11) Aliquot cell in pre chilled vials and freeze liquid nitrogen.

SOB medium: 2% bacto tryptone 0.5% yeast extract 10mM NaCl 2.5mM KCl 10mM MgCl2 10mM MgSO4 pH6.7-7

TB buffer (50ml) 10mM Pipes ~ 0.15g 15mM CaCl2 ~ 0.11g 250mM KCl ~ 0.93g à adjust pH to 6.7 with NaOH 55mM MnCl2 ~ 0.55g