Team:Bielefeld-CeBiTec/Media

iGEM Bielefeld 2015


Media & Buffers

All of our favorite recipes.

Media

LB

  • For 1 liter LB:
    • 20 g LB powder (Merck)
    • Fill the bottle with deionized H2O

  • For 1 L LB-plates:
    • 18 g LB powder (Merck)
    • 16 g Select Agar (Invitrogen)
    • Fill the bottle with deionized H2O

SOC

  • Add the following components for 900 mL of distilled H2O:
    • 20 g Trypton
    • 5 g Bacto Yeast Extract
    • 2 mL of 5 M NaCl
    • 2.5 mL of 1 M KCl
    • 10 mL of 1 M MgMgCl2
    • 10 mL of 1 M MgSO4
    • 20 mL of 1 M glucose

2xYT+P

  • For 1 L, combine
    • 16 g tryptone
    • 10 g yeast extract
    • 5 g NaCl
    • 22 mL KH2PO4
    • 40 mL K2HPO4
  • dissolve in water and titrate to pH = 7.0 with NaOH, autoclave

Buffers

50X TAE Stock Solution

  • For each litre of solution:
    • 242 g TRIS Base (MW=121.1)
    • 57.1 ml Glacial Acetic Acid
    • 100 ml 0.5 M EDTA

  • mix Tris with stir bar to dissolve in about 600 ml of ddH2O.
  • add the EDTA and Acetic Acid.
  • bring final volume to 1 l with ddH20.
  • store at room temperature.

  • Note: Final (1x) working concentration:
    • 0.04 M Tris - Acetate
    • 0.001 M EDTA

Lysis and binding buffer for purification of LacI

  • For each 200 ml of solution:
    • 5.844 g NaCl
    • 0.272 g imidazole
    • 5.8 ml of 86 % (v/v) glycerol
    • 0.406 g MgCl2
    • 0.2 g lysozyme

  • Add all these ingredients to a 50 mM sodium phosphate buffer (pH 8.0). Prior to use add DTT to a final concentration of 1 mM and tween 20 to a final concentration of 0.1 % (v/v).
  • store at room temperature.

  • Note: Final (1x) working concentration:
    • 50 mM Sodiumphosphate
    • 500 mM NaCl
    • 20 mM imidazole
    • 2.5 % glycerol
    • 1 mM DTT
    • 10 mM MgCl2
    • 0.1 % (v/v) tween 20
    • 1 mg/mL lysozyme

Equilibration buffer for LacI purification

  • For each litre of solution:
    • 29.22 g NaCl
    • 1.36 g imidazole
    • 29 ml of 86 % (v/v) glycerol

  • Add all these ingredients to a 50 mM sodium phosphate buffer (pH 8.0). Prior to use add DTT to a final concentration of 1 mM.
  • store at room temperature.

  • Note: Final (1x) working concentration:
    • 50 mM sodium phosphate
    • 500 mM NaCl
    • 20 mM imidazole
    • 2.5 % (v/v) glycerol
    • 1 mM DTT

Elution buffer for purification of LacI

  • For each litre of solution:
    • 17.53 g NaCl
    • 13.6 g imidazole

  • Add all these ingredients to a 20 mM sodium phosphate buffer (pH 7.4).
  • store at room temperature.

  • Note: Final (1x) working concentration:
    • 20 mM sodium phosphate
    • 300 mM NaCl
    • 200 mM imidazole

Buffer for dialysis of LacI/ Kpi wash buffer for PRIA

  • For each litre of solution:
    • 50 g glucose

  • Add the glucose to a 200 mM potassium phosphate buffer (pH 7.6). Prior to usage add DTT to a final concentration of 1 mM
  • store at room temperature.

  • Note: Final (1x) working concentration:
    • 200 mM potassium phosphate
    • 5 % (w/v) glucose
    • 1 mM DTT

Binding buffer for PRIA

  • 10 mM TRIS-HCl
  • Adjust pH 8 with hydrogen chloride.
  • Add 1 mM DTT to the buffer.
  • Add KCl or NaCl as needed.
  • For the optimal binding buffer 500 mM KCl is added.

HEPES (storage of proteins)

  • 50 mM HEPES
  • Adjust pH 7.2 with hydrogen chloride.

EMSA running buffer

  • 20 mM Na2HPO4, pH 8

EMSA buffer for reactions

  • 100 mM sodium phosphate buffer, pH 8.0
  • 375 mM KCl
  • 25% (w/v) glycine

Buffer for CBD-purification

  • 20 mM Na2HPO4*2 H2
  • 1.5 M NaCl
  • 1 mM EDTA
  • Dissolve all substances in water.
  • Adjust pH 8 with phosphoric acid.

Antibody stripping buffer

  • 0.2 M glycine
  • 0.05% (v/v) Tween 20
  • Adjust pH 2.5 with hydrogen chloride.

Blocking solution (DNA immobilization)

  • 0.1 M TRIS-HCl, pH 9.0
  • 50 mM ethanolamine

Blocking solution (unspecific protein interaction)

  • 1x PBS
  • 3% (w/v) milk powder solution

Buffer for annealing of oligonucleotides (PRIA)

  • 50 mM sodium phosphate, pH 8.5

Buffers and stock solutions for CFPS

Buffers

  • S30A buffer is prepared according to Sun et al. 2013 and contains
    • 14 mM Mg-glutamate
    • 60 mM K-glutamate
    • 50 mM TRIS
    at pH = 7.7 (with acetic acid), autoclaved, stored at 4 °C. Right before use, DTT is added to 2 mM final concentration.
  • S30 buffer (buffer A from Kwon and Jewett 2015) with lower TRIS concentration
    • 14 mM Mg-glutamate
    • 60 mM K-glutamate
    • 10 mM TRIS
    at pH = 8.2 (with acetic acid), autoclaved, stored at 4 °C. Right before use, DTT is added to 2 mM final concentration.

Stock solutions

  • 1.5 M Spermidine and 1 M putrescine solutions are prepared according to supporting material from Yang et al. 2012a. Briefly, a total of 4.59 mL of autoclaved ddH2O is added in small steps to 1 g of spermidine in a bottle. After each step, the bottle is mixed vigorously and the liquid is poured into a beaker where it is continously stirred. After rinsing out the whole bottle, the homogenous solution is aliquoted, flash freezed and stored at -80 °C. The procedure is similar for putrescine, though volumes differ.
  • NTP mixture is prepared from ATP, CTP, GTP and UTP stock solutions (100 mM) from Thermo Scientific. 20x NTP premix contains 30 mM of ATP and GTP, 18 mM CTP and UTP.
  • 330 mM Phosphoenolpyruvate (PEP) stock solution is prepared and pH is adjusted with 5 M KOH according to supporting material from Yang et al. 2012a

Other

10x running buffer for SDS-PAGE

  • 250 mM TRIS-HCl
  • 1.92 M Glycine
  • 1 % (w/v) SDS
  • pH 8.3

Staining solution for SDS-PAGE

  • 2 g/L Coomassie Brilliant Blue R250
  • 0.5 g/L Coomassie Brilliant Blue G250
  • 25 % (v/v) Isopropanol
  • 10 % (v/v) Acetic acid

6x PBJR for preparation of SDS-PAGE samples

  • 7 ml Tris-HCl
  • 3 ml 37 % (v/v) glycerol
  • 0.5 M SDS
  • 0.93 g dithiothreitol
  • 1.2 mg bromophenol blue

Stacking gel for SDS-PAGE

  • 1.3 mL rotiphorese-gel 30
  • 6.1 mL ddH2O
  • 2.5 mL 0.5 M TRIS-HCl, pH 6.8
  • 0.1 mL 10% (w/v) SDS
  • Add 50 µl 10 % (w/v) ammonium persulfate and 5 µl 99 % (v/v) TEMED to each aliquote and mix.

Resolving gel for SDS-PAGE

  • 4.7 mL rotiphorese-gel 30
  • 2.7 mL ddH2O
  • 2.5 mL 0.5 M TRIS-HCl, pH 6.8
  • 0.1 mL 10 % (w/v) SDS
  • Add 50 µl 10 % ammonium persulfate (w/v) and 10 µl TEMED and mix.
  • Pour the solution quickly into the gel casting form. Leave about two centimeters below the bottom of the comb for the stacking gel.

SDS destaining solution

  • For 1 liter destaining solution:
    • 450 mL ethanol
    • 100 mL acetic acid
    • Fill the bottle with deionized H2O

MALDI standard solution

  • Contains angiotensin II, substance P, adrenocorticotropic hormones, clip 1-17 and clip 18-39
  • Dissolve 1:25 with water.

Trypsin solution

  • 1 µL trypsin
  • 14 µL 10 mM NH4HCO3
  • For this solution solubilize lyophilized trypsin in 200 µL of provided buffer and activate trypsin for 15 minutes at 30 °C. For further use it can be stored at -20 °C

HCCA Matrix for MALDI

  • 711 µL 100% acetonitrile
  • 37 µL 0.1% (v/v) TFA
  • 36 µL HCCA stock solution
  • 8 µL 10% (v/v) TFA
  • 8 µL 100 mM NH4H2PO4

PDITC solution for activation of paper

  • p-phenylen-diisothiocyanate
  • Dissolve in pure ethanol or acetic acid or dried DMSO.

Primers