LB Agar

• Add:
  • 10 g/L NaCl
  • 10 g/L Bacto-Tryptone
  • 5 g/L Bacto-Yeast Extract
  • 15 g/L Bacto-Agar
  • ddH2O
  to a sterile 1L flask

• autoclave
• (add antibiotic when the agar becomes visible in the liquid, at about 45°C; shake gently to avoid bubbles)
• pour into Petri plates and close them immediately
• 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

• Prepare SOB and add 20 ml of 1M glucose (prepare it dissolving 3.6 g of glucose in 20 ml of ddH2O).

M9 supplemented with glycerol (M9gly)

For 1L of medium, add:

• 734 ml of autoclaved (and cooled to Tamb, N.B. consider evaporation during autoclaving) ddH2O with a magnetic stirrer inside the bottle
• 5 ml of autoclaved 80% glycerol as carbon source
• 100 ul of autoclaved or filtered (0.2um) CaCl2 1 M
• 20 ml of 10% autoclaved casamino acids (dissolve 50 g in 500 ml = 10% stock)
• 34 ml of filtered (0.2um) thiamine hydrochloride MW=337.27g/mol (340 mg in 34 ml) (keep in mind it is photosensitive)
• 2 ml of autoclaved MgSO4 1 M
• 200 ml of autoclaved M9 salts 5x (dissolve 56.4 g in 1 liter ddH2O = 5x stock)
• shake the ddH2O with the magnetic stirrer and start adding the other solutions in sterility (each solution must be completely dissolved!) in the order listed above.
• add antibiotic if needed
• store at +4°C, protected from light

NOTE:

• M9 salts 5x, 10% casamino acids, MgSO4 1 M and CaCl2 1 M can be stored at +4°C.
• glycerol 80% can be stored at room temperature.
• thiamine hydrochloride (LIGHT SENSITIVE) is one-shot and must be prepared each time (keep in mind you loose some volume during filtration)

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 2000 rpm, 25°C for 10 min
• take 750ul 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

E. coli competent cells preparation

H. Inoue et al. (1990), High efficiency transformation of Escherichia coli with plasmids, Gene 96 23-28.

DAY1

inoculum 5-8 ul from -80°C stock in 5 ml of LB (37°C, 220 rpm ON);

DAY2

dilution 1:1000 in SOB (flask, 18-25°C, 220 rpm ON);

DAY3

pre-chill centrifuge at 4°C;

prepare TB (prepare 50 ml every 125 ml of SOB):

• 15mM CaCl2
• 250mM KCl
• 10mM (3 g/L) Pipes
• adjust pH at 6.7 with KOH
• 55mM (8.9 g/L) MnCl2
• filter (0.2 um) the solution and chill in 50 ml

put the flask in ice when the culture reaches OD600=~0.05 (1mm pathlength – NanoDrop);

aliquot in pre-chilled 50 ml falcon tubes;

centrifuge at 2500g (4400rpm), 4°C, 10 min;

ICE: discard, resuspend in 40 ml of TB each 125 ml SOB, centrifuge as before;

ICE: discard, resuspend in 10 ml of TB each 125 ml SOB, add 700ul DMSO;

ICE: aliquot 100ul in pre-chilled 0.5ml tubes;

put in -80°C freezer;

ALWAYS TEST THE EFFICIENCY IN [CFU/ug] UNITS: transform 100ul of competent cells with 4ng of DNA and 100ul of competent cells without DNA (add 1ul of ddH2O), then plate on proper LB agar plates.

This protocol has shown to work with:

• DH5alpha (10^8 with 100ul of cells);
• TOP10 (5*10^7 with 100ul of cells);
• BW20767 (10^3 with 100ul of cells);
• DB3.1 (5*10^4 with 100ul of cells);

J. Sambrook, E.F. Fritsch, T. Maniatis (1989)

• DAY1
  • Inoculum 5-8 ul from -80°C stock in 5 ml of LB (37°C, 220 rpm ON).
• DAY2
  • Dilution 1:500 in LB (flask, 30-37°C, 220 rpm), monitor OD600 until it reaches 0.04 (1mm pathlength – NanoDrop, it should take from 3 to 5 hours);
  • prepare the proper amount of 50 ml tubes in ice and pre-chill the centrifuge;
  • when the culture reaches the right OD600, aliquot the culture in the pre-chilled tubes;
  • centrifuge (4000rpm, 4°C, 10min) and discard the supernatant;
  • for each 50 ml of culture, add 30 ml of MgCl2-CaCl2 solution (Buffer1) and resuspend the pellet;
  • centrifuge (4000rpm, 4°C, 10min) and discard the supernatant;
  • for each 50 ml of the original culture, add 2 ml of CaCl2 solution (Buffer2) and resuspend the pellet;
  • aliquot in 0.5 ml tubes and store at -80°C.