Team:Bielefeld-CeBiTec/Notebook/Media

From 2014.igem.org


Media

Media
  • For 1 liter LB:
    • 20 g LB (Lenox Bruth) powder
    • Fulfill the bottle with deionized H2O

  • For 1 liter LB-plates:
    • 18 g LB (Lenox Broth) powder
    • 16 g Select Agar
    • Fulfill the bottle with deionized H2O
  • 12 g Tryptone
  • 24 g yeast extract
  • 4 ml Glycerol
  • dissolve the solutes in 900 ml of deionized H2O
  • Salt Solution:
    • KH2PO4 (0,17M) equals 2.31 g per 100 milliliters
    • K2HPO4 (0,72 M) equals 12.54 g per 100 millilters
    • dissolve the solutes in 100 ml of deionized H2O and mix them with the other media components to get a total volume of 1 litre
  • 15 g Agar-Agar per liter (for plates)
  • To prepare 1 liter of M9 minimal medium add the following components to 836.7 ml sterile distilled water. To avoid precipitation, start with CaCl2 and mix the solution thoroughly after addition of a component.
  • 100 ml 10 X M9 salt solution
  • 50 ml 20 X carbon source
  • 1 ml 1000 X trace element solution
  • 1 ml autoclaved 1 M MgSO4
  • 0.3 ml autoclaved 1 M CaCl2
  • 1 ml filter sterilized 1 g/l biotin
  • 1 ml filter sterilized 1 g/l thiamin
  • 75.2 g Na2HPO4 x 2H2O
  • 30 g KH2PO4
  • 5 g NaCl
  • 5 g NH4Cl
  • Dissolve the salts in 800 ml water and adjust the pH to 7.2 with NaOH. Add water to a final volume of 1 l and autoclave for 15 minutes at 121 °C.
  • 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 MgCl2
    • 10 ml of 1 M MgSO4
    • 20 ml of 1 M glucose


Buffer
  • All buffers with pH 7.4 - 7.6
NameSodium phosphateNaClImidazolEDTA
Binding Buffer20 mM500 mM5 mM0 mM
Elution Buffer 120 mM500 mM40 mM0 mM
Elution Buffer 220 mM500 mM60 mM0 mM
Elution Buffer 320 mM500 mM100 mM0 mM
Elution Buffer 420 mM500 mM300 mM0 mM
Elution Buffer 520 mM500 mM500 mM0 mM
Stripping Buffer20 mM500 mM0 mM50 mM
  • 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
  • For each litre of solution:
    • 242 g Tris Base (MW=121.1)
    • 57.1 ml Glacial Acetic Acid
    • 10 ml 0.5 M EDTA

  • mix Tris with stir bar to dissolve in about 600 ml of ddH2O.
  • add the EDTA and Acetic Acid, pH to 8.0.
  • bring final volume to 1 l with ddH2O.
  • store at room temperature.

  • Note: Final (1x) working concentration:
    • 0.04 M Tris - Acetate
    • 0.0001 M EDTA
  • 1 l of 50x TAE buffer
  • 242.48 g Tris
  • 41.02 g sodium acetate
  • 18.612 g EDTA
  • Adjust pH to 7.8 with acetic acid
  • Solve in dH2O
  • Dilute 20 ml 50x stock in 1 l dH2O for 1x Buffer for PAGE
  • 292.243g/mol 1mM EDTA
  • 0.025 g 0.05% (w/v) BPB
  • 0.025 g 0.05% (w/v) Xylene Cyanol
  • Solve in H2O
  • Adjust color to green with HCl
  • Dilute with glycerol to 50:50
  • For 50 ml:
    • 5 g PEG 8000
    • 1.5 ml 1M MgCl2 (or 0.30 g MgCl2*6H2O)
    • 2.5 ml DMSO
    • Add LB to 50 ml
    • Store at 4°C or -20°C
  • For 1 l:
    • 3 g Tris
    • 14.4 g Glycine
    • 1 g SDS
  • For 10 ml (6x buffer):
    • 7 ml Tris-HCl
    • 3 ml Glycerol (37 %)
    • 0.5 M SDS
    • 0.93 g DTT
    • 1.2 mg bromphenol blue (BPB)
    • 2.5 g/l Coomassie Brilliant Blue R250
    • 10 % (v/v) Acetic Acid
    • 25 % (v/v) Isopropyl alcohol
    • 100 mM Tris-HCl, pH 6,8
    • 1 M ß-Mercaptoethanol
    • 6 % SDS
    • 12 % Glycerol
    • 0.2 % bromphenol blue (BPB)
  • 3 ml of 1M Tris-HCl (pH 7.5)
  • 150 µl of 2 M MgCl2
  • 60 µl of 100 mM dGTP
  • 60 µl of 100 mM dATP
  • 60 µl of 100 mM dTTP
  • 60 µl of 100 mM dCTP
  • 300 µl of 1 M DTT
  • 1.5 g PEG-8000
  • 300 µl of 100 mM NAD
Cell Fractioning Buffer 1
  • 0.2 M Tris-HCl (pH 8.0)
  • 200 g L-1 sucrose
  • 0.1 M EDTA

Cell Fractioning Buffer 2
  • 0.01 M Tris-HCl (pH 8.0)
  • 0.005 MgSO4
  • 0.2% SDS
  • 1% Triton X-100
1X PBS (Phosphate buffered saline)
  • 137 mM NaCl
  • 2.7 mM KCl
  • 10 mM Na2HPO4
  • 2 mM KH2PO4
  • adjust pH to 7.4 with HCl
Buffer for the cathode space (if cell free)
  • 50 mM KH2PO4
  • 5 mM NaCl
  • 100 µM Mediator

Buffer for the anode space
  • 50 mM KH2PO4
  • 100 mM NaCl
  • Adjust the pH of both buffers to 7.2 with NaOH.