Media & Antibiotics

LB

• Add:
  • 10 g/L NaCl
  • 10 g/L Bacto-Tryptone
  • 0.5 g/L Bacto-Yeast Extract
  • ddH2O

to a sterile pyrex bottle

• autoclave
• (add antibiotic when it reaches ~45°C)
• store at +4°C

LB Agar

• Add:
  • 10 g/L NaCl
  • 10 g/L Bacto-Tryptone
  • 0.5 g/L Bacto-Yeast Extract
  • 15 g/L Bacto-Agar
  • ddH2O

to a sterile 1L flask

• autoclave
• (add antibiotic when it reaches ~45°C, shake gently to avoid bubbles)
• pour into Petri plates
• let them polymerize for ~2-3h
• invert plates and wrap them with aluminium foil and store at +4°C

SOB

• Add:
  • 5 g/L Bacto-Yeast Extract
  • 20 g/L Bacto-Tryptone
  • 10mM NaCl
  • 2.5mM KCl
  • 10mM MgSO4
  • 10mM MgCl2

to a sterile pyrex bottle

• (optional: check that pH is ~6.8, otherwise adjust with NaOH)
• autoclave
• (add antibiotic when it reaches ~45°C)
• store at +4°C

SOC

• SOB+20mM (3.6 g/L) of glucose (add filter-sterilized (0.2um) glucose to autoclaved SOB).

M9 supplemented with glycerol (M9gly)

For 1L of medium, add:

• 716 ml of autoclaved (and cooled to Tamb) ddH2O
• 200 ul of autoclaved or filtered (0.2um) CaCl2 0.5 M
• 200 ml of autoclaved M9 salts 5x (dissolve 56.4 g in 1 liter ddH2O = 5x stock)
• 34 ml of filtered (0.2um) thiamine hydrochloride MW=337.27g/mol (340 mg in 34 ml)
• 20 ml of autoclaved MgSO4 0.1 M
• 20 ml of 10% autoclaved casamino acids (dissolve 50 g in 500 ml = 10% stock)
• 10 ml of autoclaved 40% glycerol as carbon source
• mix all the solutions in sterility (each solution must be completely dissolved!)
• (add antibiotic)
• store at +4°C, protected from light

NOTE:

• M9 salts 5x
• 10% casamino acids

can be stored at +4°C

• MgSO4 0.1 M
• CaCl2 0.5 M
• glycerol 40%

can be stored at room temperature or +4°C

• thiamine hydrochloride (LIGHT SENSITIVE) is one-shot and must be prepared each time

Antibiotics

Stocks at -20°C freezer:

• Ampicillin 100 mg/ml (in water)
• Kanamycin 50 mg/ml (in water)
• Chloramphenicol 34 mg/ml (in 100% ethanol)

These stocks are 1000x for high copy number plasmids. For low copy number plasmids, you should use these final concentrations in media:

• Ampicillin 50 ug/ml
• Kanamycin 20 ug/ml
• Chloramphenicol 12.5 ug/ml

E. coli transformation

Transforming home-made competent cells

• heat ligation at 65°C to inactivate T4 ligase
• thaw in ice a vial of TOP10 competent cells stored at -80°C
• incubate a selective LB agar plate at 37°C
• pipet 800ul of LB (without antibiotic) in a 15ml falcon tube and incubate it at 37°C
• heat the water bath at 42°C

• add 1 ul (~3ng of DNA vector) of ligation to 100ul of thawed TOP10
• add parafilm and incubate in ice for 30 min
• heat shock at 42°C for 1 min
• incubate in ice for 2 min
• transfer transformed bacteria to 800ul of pre-warmed LB
• incubate at 37°C, 220 rpm for 1 h
• centrifuge at 1200 rpm, 25°C for 10 min
• take 650ul of supernatant and resuspend the pellet in the remaining LB (~150ul)
• plate the entire culture and incubate the plate at 37°C overnight

Variants:

• if you transform a miniprep, add less than 3 ng in order to have single colonies
• if you use another home-made competent strain, the protocol is the same but you should consider the transformation efficiency to add a proper amount of DNA
• if you use commercial Invitrogen TOP10 the protocol changes and it is reported below.

Transforming commercial competent cells

(according to manufacturer’s protocol)

• heat ligation at 65°C to inactivate T4 ligase
• thaw in ice a vial of TOP10 competent cells stored at -80°C (one vial contains 50ul of cells)
• incubate a selective LB agar plate at 37°C
• heat the water bath at 42°C

• dilute the ligation 1:50 (or 1:100) in ddH2O, in order to have less than 100pg/ul
• add 1 ul of ligation (or less than 100pg of miniprepped DNA) to 25 or 50ul of thawed TOP10
• add parafilm and incubate in ice for 10 min
• heat shock at 42°C for 1 min
• incubate in ice for 2 min
• add 250ul of SOC medium
• incubate at 37°C, 220 rpm for 1 h
• plate 150ul of the culture and incubate the plate at 37°C overnight
• the remaining 150ul can be stored at +4°C