From 2010.igem.org

Revision as of 13:43, 17 June 2010 by Baren (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

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