|
|
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
|
"
