Team:Bielefeld-CeBiTec/Notebook/Media

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(Difference between revisions)
Line 437: Line 437:
     <li>7 mL Tris-HCl</li>
     <li>7 mL Tris-HCl</li>
     <li>3 mL Glycerol (37 &#37;)</li>
     <li>3 mL Glycerol (37 &#37;)</li>
-
     <li>0.5 M SDS (1 g)</li>
+
     <li>0.5 M SDS</li>
     <li>0.93 g DTT</li>
     <li>0.93 g DTT</li>
     <li>1.2 mg bromphenol blue (BPB)</li>
     <li>1.2 mg bromphenol blue (BPB)</li>

Revision as of 19:29, 13 October 2014


Media

Media
LB medium

LB medium

  • For 1 liter LB:
    • 20g 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
Terrific Broth (TB)

Terrific broth (TB)

  • 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)
M9 medium

M9 medium

  • 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
M9 salt stock solution

M9 salt stock solution

  • 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.
SOC-medium

SOC-medium

  • 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
His Trap Buffer

His Trap 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
50X TAE Stock Solution

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
50X Modified TAE Stock Solution

50X Modified TAE Stock Solution

  • 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
TAE buffer

TAE buffer

  • 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 1L dH2O for 1x Buffer for PAGE
Loading buffer

Loading Buffer

  • 292.243g/mol 1mM EDTA
  • 0.025g 0.05% (w/v) BPB
  • 0.025g 0.05% (w/v) Xylene Cyanol
  • Solve in H2O
  • Adjust color to green with HCl
  • Dilute with glycerol to 50:50
TSS buffer

TSS Buffer

  • For 50 mL:
    • 5g PEG 8000
    • 1.5 mL 1M MgCl2 (or 0.30g MgCl2*6H2O)
    • 2.5 mL DMSO
    • Add LB to 50 mL
    • Store at 4°C or -20°C
SDS-PAGE running buffer

SDS-PAGE running buffer

  • For 1 L:
    • 3 g Tris
    • 14.4 g Glycine
    • 1 g SDS
PBJR buffer

PBJR buffer

  • 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)
Colloidal Coomassie Brilliant Blue staining solution

Colloidal Coomassie Brilliant Blue staining solution

    • 2.5 g/L Coomassie Brilliant Blue R250
    • 10 % (v/v) Acetic Acid
    • 25 % (v/v) Isopropyl alcohol
Fast Cell Lysis sample buffer

Fast Cell Lysis sample buffer

    • 100 mM Tris-HCl, pH 6,8
    • 1 M ß-Mercaptoethanol
    • 6 % SDS
    • 12 % Glycerol
    • 0.2 % bromphenol blue (BPB)
5x isothermal reaction buffer for Gibson Assembly

5x isothermal reaction buffer for Gibson Assembly

  • 3mL 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 for Cold Osmotic Shock

Cell Fractioning Buffer for Cold Osmotic Shock

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
PBS Buffer

PBS Buffer

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

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