Team:Bielefeld-CeBiTec/Experiments/Media






Media and Buffers

Media and buffers that we used

Media

LB

  • For 1 L LB:
    • 20 g LB powder
    • Fill the bottle with deionized H2O

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

SOC

  • For 10 ml SOC:
    • 9.5 ml Trypton/Bacto Yeast Extract
    • 100 μl of 5 M NaCl
    • 25 μl of 1 M KCl
    • 100 μl of 1 M MgCl2
    • 100 μl of 1 M MgSO4
    • 200 μl of 1 M glucose

Buffers

50X TAE Stock Solution

  • For 1 L:
    • 242 g TRIS Base (MW=121.1)
    • 57.1 ml Glacial Acetic Acid
    • 100 ml of 0.5 M EDTA

  • dissolve TRIS in 600 ml of ddH2O.
  • add EDTA and Acetic Acid.
  • fill with ddH2O to a final volume of 1 L.
  • store at room temperature.

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

5X TBE Stock Solution

  • For 1 L:
    • 54 g TRIS Base (MW=121.1)
    • 27.5 g boric acid
    • 20 ml of 0.5 M EDTA (pH 8.0)

  • fill with H2O to a final volume of 1 L.
  • store at room temperature.

  • Note: Final (0.5x) working solution:
    • 45 mM TRIS - borate
    • 1 mM EDTA

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

Impact lysis buffer

  • For 1 L:
    • 1 ml Triton X-100
    • 200 μl PMSF
    • 100 μl - 1 ml TCEP

  • for PMSF (protease-inhibitor): prepare 100 mM stock solution; dissolve in DMSO; freezee.
  • for TCEP (stabilizer): prepare 1 M stock solution; dissolve in H2O.
  • dissolve all substances in column buffer (low salt).
  • store at temperature 4°.

Impact column buffer

  • For 1 L:
    • 3.6 g of 20 mM Na2HPO4 ·H2O
    • 58.44 g of 100 mM NaCl
    • 10 ml of 1 mM EDTA

  • for EDTA: prepare 0.1 M stock solution; dissolve in H2O.
  • dissolve all substaces in H2O.
  • adjust to pH 8 with phosphoric acid.
  • store at temperature 4°.

Impact cleavage buffer

  • For 1 L:
    • 3.6 g of 20 mM Na2HPO4 · H2O
    • 29 g of 50 mM NaCl
    • 10 ml of 1 mM EDTA
    • 0.93 g of 50 mM DTT (dark!)

  • after adding DTT hold solution dark!
  • add 6 ml H2O to DTT.
  • dissolve all substances in H2O.
  • adjust to pH 7-9 with phosphoric acid.
  • store at 4°.

Impact protein-wash buffer

  • For 1 L:
    • 3.6 g of 20 mM Na2HPO4 · H2O
    • 0.58 g of 10 mM NaCl
    • 10 ml of 1 mM EDTA

Osmotic shock buffer

  • For 0.5 ml (for 1 ml culture):
    • 200 mM TRIS base, pH 7.5
    • 1 mM EDTA
    • 20 % sucrose
    • 500 μg/ml lysozym

Phosphat buffer pH 7, 100 mM

  • For 100 ml:
    • 61.5 ml of 1 M K2HPO4
    • 38.5 ml of 1 M KH2PO4

PEG NaCl for phagemid display

  • For 1 L:
    • 1 M PEG
    • 1 M NaCl

PBST for phagemid display

Salt Concentration (mmol/L) Concentration (g/L)
NaCl 137 8.0
KCl 2.7 0.2
Na2HPO4 10 1.42
KH2PO4 1.8 0.24
  • Adding 0.1 % Tween

PBS for phagemid display

Salt Concentration (mmol/L) Concentration (g/L)
NaCl 137 8.0
KCl 2.7 0.2
Na2HPO4 10 1.42
KH2PO4 1.8 0.24

NaHCO3 for coating proteins for phagemid display

  • For 1 L:
    • 1 M NaHCO3

Glycine-HCl for phagemid display

  • For 1 L:
    • 0.1 M Glycine-HCL

  • Adjust pH to 2.7

Tris for phagemid display

  • For 1 L:
    • 1 M Tris

  • Adjust pH to 9

Other

10x running buffer for SDS-PAGE

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

1x running buffer for native SDS-PAGE

  • 25 mM TRIS
  • 192 mM Glycine
  • pH 8.3

6x PBJR for preparation of SDS-PAGE samples

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

2x Sample buffer for preparing SDS-PAGE samples

  • 62.5 mM Tris-HCl, pH 6.8
  • 25 % glycerol
  • 1 % bromophenol blue

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

Stacking gel for SDS-PAGE

  • 1.3 ml rotiphorese-gel 30
  • 6.1 ml ddH2O
  • 2.5 ml of 0.5 M TRIS-HCl, pH 6.8
  • 0.1 ml of 10% (w/v) SDS

  • Add 50 μl 10 % (w/v) ammonium persulfate and 5 μl 99 % (v/v) TEMED to each aliquote, mix and spill immediately.
  • Pour the solution quickly into the gel casting form and put the comb in.

Stacking gel for native SDS-PAGE

  • For 5 ml:
    • 0.67 ml rotiphorese-gel 30%, 0.8 % w/v
    • 4.275 ml of 0.375 M TRIS-HCl, pH 8.8

  • Add 0.05 μl 10 % (w/v) ammonium persulfate and 5 μl 99 % (v/v) TEMED to each aliquote and quickly mix.
  • Pour the solution quickly into the gel casting form and put the comb in.

Resolving gel for SDS-PAGE

  • 4.7 ml rotiphorese-gel 30
  • 2.7 ml ddH2O
  • 2.5 ml of 0.5 M TRIS-HCl, pH 6.8
  • 0.1 ml of 10 % (w/v) SDS

  • Add 50 μl of 10 % ammonium persulfate (w/v) and 10 μl TEMED and quickly mix.
  • Pour the solution quickly into the gel casting form. Leave about two centimeters below the bottom of the comb for the stacking gel.

Resolving gel for native SDS-PAGE

  • For a 10 ml native gel, 12% acrylamid:
    • 4.0 ml rotiphorese-gel 30%, 0.8% w/v
    • 5.89 ml of 0.375 M TRIS-HCl, pH 8.8

  • Add 100 μl of 10 % ammonium persulfate (w/v) and 10 μl TEMED and quickly 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