Team:Wageningen UR/Project/ProtocolsProj1

Protocols

Media

LB

Ingredients:

10 g Bacto-tryptone

5 g yeast extract

10 g NaCl

SOB

Ingredients

0.5% (w/v) yeast extract

2% (w/v) tryptone

10 mM NaCl

2.5 mM KCl

20 mM MgSO4

Per liter:

5 g yeast extract

20 g tryptone

0.584 g NaCl

0.186 g KCl

2.4 g MgSO4

SOC

Ingredients

SOB

20 mM glucose

CCMB80 buffer for preparation of chemically competent cells

Materials

Detergent-free, sterile glassware and plasticware

Table-top OD600nm spectrophotometer

SOB

CCMB80 buffer

10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)

80 mM CaCl2.2H2O (11.8 g/L)

20 mM MnCl2.4H2O (4.0 g/L)

10 mM MgCl2.6H2O (2.0 g/L)

10% glycerol (100 ml/L)

Adjust pH DOWN to 6.4 with 0.1N HCl if necessary

Adjusting pH up will precipitate manganese dioxide from Mn containing solutions

Filter sterilize and store at 4°C

Slight dark precipitate appears not to affect its function