Media

LB Medium

For 500 mL:

• 5 g tryptone
• 2.5 g yeast extract
• 5 g NaCl
• Add 475 mL water, dissolve solids, add 0.1 mL 5 M NaOH, make to 500 mL with water. Autoclave and store at room temperature. Refrigerate after opening.

Notes

• For agar plates, add 15 g agar prior to autoclaving
• For ampicillin plates, add 1 mL sterile 50 mg/mL ampicillin after cooling to 50°C

TB Overexpression Medium

For 1000 mL:

• Nutrient solution
  • 12 g tryptone
  • 24 g yeast extract
  • 8 mL 50% glycerol
  • Make to 900 mL and autoclave
• Buffer solution
  • 2.31 g KH2PO4
  • 12.54 g K2HPO4
  • Make to 100 mL and autoclave.
• Cool to 55 °C or less, combine, and add any antibiotics desired.

Notes

• Store and autoclave nutrient and buffer solutions separately
• Combine only immediately prior to usage

SOB Medium

For 500 mL:

• Nutrient solution
  • 10 g tryptone
  • 2.5 g yeast extract
  • 0.25 g NaCl
  • 0.95 g KCl
  • 0.5 g MgCl2
  • 0.95 g MgSO4
  • Make to 500 mL and mix to dissolve/suspend solids
  • Check pH with pH paper and adjust to approx. pH 7 with NaOH (usually not necessary)
• Autoclave

Notes

To make SOC medium, add sterile 2M glucose to make a final concentration of 20 mM (100X dilution)

Buffers

1 M Tris

• 121.1g Tris base
• Dissolve in 800 mL water, adjust pH to desired value (pH 7.4, 7.8, or 8.0) with HCl, make to 1000 mL with water
• Sterilize by autoclaving.

0.5 M EDTA (pH 8.0)

• 186.1 g disodium EDTA
• Dissolve in 800 mL of water, adjust pH to 8.0 with NaOH, make to 1000 mL
• Sterilize by autoclaving.

TE Buffer

• 10 mL 1 M Tris (desired pH)
• 2 mL 0.5 M EDTA (pH 8.0)
• Make to 1000 mL with water.

TAE Buffer (50 x)

• 242 g Tris
• 57.1 mL acetic acid
• 100 mL 0.5 M EDTA (pH 8.0)
• Make to 1000 mL with water.

Notes

• 1 x composition is 40 mM Tris-acetate-1 mM EDTA

TBE Buffer (5 x)

• 54 g Tris
• 27.5 g boric acid
• 20 mL 0.5 M EDTA (pH 8.0)
• Make to 1000 mL with water.

Notes

• 1 x composition is: 90 mM Tris-borate-2 mM EDTA

Agarose Gel Loading Buffer (6 x)

• 0.25% (w/v) bromophenol blue
• 40% (w/v) sucrose
• Make to volume with water
• Store at 4 °C.

Transformation Buffer (TSS, 2 x)

• 5 g PEG 8000
• 0.25 g MgSO₄ • 7 H₂O
• Add 22.5 mL LB, adjust pH to 6.5-6.8 if necessary.
• Sterilize by filtration
• Add 2.5 mL DMSO.
• Store at 4 °C.

Notes

• Composition is 20% PEG, 40 mM Mg²⁺, 10% DMSO.

Reagents

dNTP mix

Our PCR protocols call for 2.5 mM (each) dNTPs.

• Thaw 10 mM (each) dNTP mix stock solution at 37 °C and spin to collect
• Dilute 4X by pipetting 10 μL dNTP stock solution into 30 μL water
• Mix thoroughly; spin to collect.
• Store at -20 °C.

10% Ammonium Persulfate

• 1 g ammonium persulfate
• Make to 10 mL with water
• Store in 0.1 mL aliquots at –20 °C.

5% Ampicillin

• 500 mg ampicillin, sodium salt
• Dissolve in 10 mL of water.
• Sterile filter and store at 4 °C.
• This solution is 500X (final 100 μg/mL)

Chloramphenicol, 20 mg/mL

• 200 mg chloramphenicol
• Dissolve in 10 mL of 100% or 95% ethanol
• Sterile filtration not necessary
• This solution is 1000X (final 20 μg/mL)

Tetracycline, 0.5 mg/mL

• 50 mg of tetracycline HCl
• Dissolve in 7 mL 100% ethanol
• Dilute with water to 10 mL
• Sterile filtration not necessary
• This solution is 100,000X for Pzt promoter induction (final 5 ng/mL)

L-arabinose, 200 mg/mL

• 2 g arabinose
• Dissolve in water to final volume of 10 mL
• Sterile filter
• Solution is 400X for arabinose promoter induction (final 0.5 mg/mL)

200 mM IPTG

• 476 mg IPTG
• Dissolve in 10 mL of water
• Sterile filter in 1 mL aliquots
• Store at –20 °C.

2% X-gal

• 20 mg X-gal
• Dissolve in 1 mL of DMF.
• Store in dark bottle at –20 °C.

1% Ethidium Bromide

• 100 mg ethidium bromide
• Dissolve in 10 mL of water
• Store in dark bottle

Phenol

• Add 0.1 g of 8-hydroxyquinoline to 100 mL of liquid phenol.
• Add 100 mL of 500 mM Tris base, stir 10 min at low speed with a stir bar, decant aqueous layer.
• Check phenol layer with pH paper to verify pH is ≥8.0, reequilibrate if necessary.
• Equilibrate similarly 2x with 100 mL of 50 mM Tris-Cl ( pH 8.0)
• Store the equilibrated mixture under an equal volume of 50 mM Tris-Cl (pH 8.0) at 4 °C in a dark bottle

Chloroform

• 24 parts chloroform
• 1 part isoamyl alcohol
• Store in a dark bottle.

5 M Sodium Hydroxide

• 263 mL concentrated (19.1 M) sodium hydroxide
• Dilute to 1000 mL with water
• Store in a plastic bottle

2 M NaOH–1 mM EDTA

• 40 mL 5 M sodium hydroxide
• 0.2 mL 0.5 M EDTA
• Dilute to 100 mL with water.
• Store in a plastic bottle.

3 M Sodium Acetate (pH 4.9)

• 408.1 g sodium acetate trihydrate
• Dissolve in a minimum volume of water, titrate to pH 4.9 with glacial acetic acid
• Make to 1000 mL with water
• Sterilize by autoclaving.