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

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

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);

E. coli strains (all in -80°C freezer)

TOP10

F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-

• source: Invitrogen
• competent cells already prepared (5*10^7 CFU/ug with100ul of cells)
• competent cells from Invitrogen available (10^9 CFU/ug with 50ul of cells)
• commonly used for cloning and expression in our lab
• they are equal to DH10B strain, whose genome is available from NCBI

NOTE: they have

• lacI wt
• cI of phi80 prophage (different from cI of lambda phage)
• Streptomycin resistance

DH5alpha

F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–

• source: Francesca Ceroni
• competent cells already prepared (10^8 CFU/ug with100ul of cells)
• commonly used for cloning

BW20767

F-, RP4-2(Km::Tn7,Tc::Mu-1), leu-163::IS10, ΔuidA3::pir+, recA1, endA1, thi-1, hsdR17, creC510

• source: Vinoo Selvarajah
• competent cells already prepared (10^3 CFU/ug with100ul of cells)
• not used for cloning

NOTE: they have

• a fully working lac operon (already tested on IPTG/X-Gal plates)
• Kan and Tet resistance (not tested)

XL1-Blue

endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F'[ ::Tn10 proAB+ lacIq Δ(lacZ)M15] hsdR17(rK- mK+)

• source: Francesca Ceroni
• competent cells never prepared
• a small stock of competent cells is available
• used for cloning

NOTE: they have lacIQ

DB3.1

F- gyrA462 endA1 glnV44 Δ(sr1-recA) mcrB mrr hsdS20(rB-, mB-) ara14 galK2 lacY1 proA2 rpsL20(Smr) xyl5 Δleu mtl1

• source: Francesca Ceroni
• competent cells already prepared (5*10^4 CFU/ug with 100ul of cells)
• used for in vivo amplification of ccdB plasmids

NOTE: they have a working lacZ, but a deleted lacY, they become slightly blue on IPTG/X-Gal plates

STBL3

F- glnV44 recA13 mcrB mrr hsdS20(rB-, mB-) ara-14 galK2 lacY1 proA2 rpsL20 xyl-5 leu mtl-1

• source: Invitrogen
• competent cells never prepared
• used for in vivo amplification of DNA with direct repeats

NOTE: they cannot be used for blue/white screening

CW2553 + pJat8

Genotype: Khlebnikov A et al. (2000), Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture, Journal of Bacteriology, Vol. 182, No. 24, p.7029-7034.

• Source: Vinoo Selvarajah
• pJat8 is Gentamycine resistant

NOTE:

• the stock of this strain has been grown without Gen
• this strain is used for araBAD inducible system

Long term bacterial glycerol stocks

• Mix 750 ul of a culture (preferably in log-phase) with 250 ul of 80% glycerol, in a 1.5ml vial
• label the vial with name, date and antibiotic resistance
• leave at -20°C for one day
• move to -80°C the day after

Plasmid digestion for BioBrick Standard Assembly

To open vectors

• a volume containing 1 ug of purified plasmid
• 2 ul (or 2.5 ul if the final volume is 25 ul) of 10X buffer H
• 1 ul of first enzyme
• 1 ul of second enzyme
• 20 ul (or 25 ul) final volume
• incubate at 37°C for 3 hours

To excide fragments

• A volume containing 1-2.5 ug of purified plasmid
• 2.5 ul of buffer H
• 1 ul of first enzyme
• 1 ul of second enzyme
• 25 ul final volume
• incubate at 37°C for 3 hours

NOTE: if you are performing a digestion for screening, 1 hour of incubation is sufficient.

Ethanol precipitation with sodium acetate

• Add 1/10 DNA solution volume of sodium acetate 3 M, pH 5.2
• Add 2.5 DNA solution volume of absolute ethanol
• Freeze at -80°C for 30 min
• Centrifuge at 13000 rpm, 4°C for 20 min
• Decant supernatant
• Add 250 µl of 70% ethanol
• Centrifuge at 13000 rpm, 4°C for 20 min
• Remove all supernatant with a pipette
• Air dry pellet until ethanol is totally removed
• Elute with 5-10 µl of ddH2O

Ligation

After the purification of two digested DNA fragments:

• add a volume containing 20-50 ng of vector
• add a volume containing

(“6” can be lowered to “2”)

• heat DNA mix at 65°C for 5 min for DNA denaturation
• add 1 ul of T4 Ligase buffer (check if ATP is completely dissolved)
• add 1 ul of T4 Ligase
• 10-20 µl final volume
• incubate at 16°C overnight
• inactivate the T4 Ligase heating at 65°C for 10 min
• then, ligation can be conserved at 4°C or can be transformed

NOTE: When the purified DNA of the insert also contains its native vector, you can perform the ligation anyway, but its antibiotic resistance must be different from the acceptor vector’s resistance in order to select correct transformants on agar plates. When doing this, you should modify the ligation protocol:

• you should use “2” or “3” instead of “6” to compute the insert mass;
• when you add the volume containing the insert mass, you must consider that the DNA quantification with NanoDrop refers to insert+NATIVE VECTOR. So, you must add:

insert volume [ul]=(insert length/(insert length+native vector length))*(Insert mass [ng])/(DNA quantification [ng/ul])