Team:Bielefeld-Germany/Labjournal/Materials

From 2013.igem.org

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<h1 style="text-color:#ff6600">Materials</h1>
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<h1 style="color:#ff6600">Materials</h1>
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<br><b><u>Biotin 500x stock solution</u></b>
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<p> - Dissolve 20 mg biotin in 100 mL of 0.05 M NaOH solution and filter sterilize</p>
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<p> - Store at 4 °C</p>
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<p> - Durable for one year</p>
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<br><b><u>TAE buffer</u></b>
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<p>For 1 L of 50 x TAE buffer you need:</p>
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<br><b><u>Cold osmotic shock buffers for the release of periplasmic protein fraction</u></b>
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<br><b style="padding-left:24px;">Cell fractionating buffer 1 (pH 8) </b>
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<p> • 0.2 M Tris </p>
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<p> • 200 g L -1 sucrose </p>
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<p> • 0.1 M EDTA</p>
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<br><b style="padding-left:24px;">Cell fractionating buffer 2.1 (pH 8)  </b>
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<p> • 0.01 M Tris </p>
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<p> • 0.005 M MgSO4 </p>
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<br><b style="padding-left:24px;">Cell fractionating buffer 2.2 (pH 8)  </b>
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<p> • 0.01 M Tris </p>
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<p> • 0.005 M MgSO4 </p>
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<p> • 1% Trition</p>
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<p> • 2% SDS</p>
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<br><b style="padding-left:24px;">Cell fractionating buffer 2.3 (pH 8) </b>
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<p> • 0.01 M Tris </p>
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<p> • 0.005 M MgSO4 </p>
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<p> • 1% Trition</p>
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<p> • 0,2% SDS</p>
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Latest revision as of 11:07, 26 September 2013







Materials


[Media]

Chloramphenicol stock solution

-Solubilize 20 mg mL-1 Chloramphenicol in 100 % Ethanol

-Store at -20 °C


DNA loading buffer

- 50 % (v/v) glycerol

- 1 mM EDTA

- 0.1 % (w/v) bromphenol blue

- Solve in ddH2O


LB medium

- 10 g Trypton

- 5 g yeast extract

- 10 g NaCl

- 12 g Agar-Agar (for plates)

- Adjust pH to 7.4


M9 medium

For 1 L M9 mineral medium you need 867 mL sterile water (for plates add 16 g Agar-Agar as well). Then add (in the following order:

- 300 µL 1 M CaCl2

1 M CaCl2 1 M CaCl2-H20 14.70 g/100 mL

- 100 mL 5x M9 salt solution

10x M9 salt solution
Na2HPO4-2H2O 75.2 g/L
KH2PO4 30 g/L
NaCl 5 g/L
NH4Cl 5 g/L

- 20 % glucose

20 % Glucose 20 % (w/v)glucose 200 g/L

For 500 mL stock solution add 100 g glucose to 440 mL water. Autoclave for 15 at 121° C

- 1 M MgSO4

MgSO4 1 M MgSO4-7H20 24.65 g/100 mL

- 1 mL biotin (1 mg/mL)

Biotin (1 mg/mL) biotin (1 mg/mL) 50 mg/50 mL

For 50 mL stock solution dissolve 50 mg biotin in 45 mL water. Add water to a final volume of 50 mL. Sterilize the solution over a 0.22-µm filter. Prepare 1 mL aliquots and store at -20°.

- 1 mL thiamin (1 mg/mL)

Thiamin (1 mg/mL) thiamin-HCl (1 mg/mL) 50 mg/50 mL

For 50 mL stock solution dissolve 50 mg thiamin-HCl in 45 mL water. Add water to a final volume of 50 mL. Sterilize the solution over a 0.22-µm filter. Prepare 1 mL aliquots and store at -20°.

- 10 mL 100x trace elements solution

100x trace elements solution EDTA 5 g/L 13.4 mM
FeCl3-6H2O 0.83 g/L 3.1 mM
ZnCl2 84 mg/L 0.62 mM
CuCl2-2H20 13 mg/L 76 µM
CoCl2-H2O 10 mg/L 42 µM
H3BO3 10 mg/L 162 µM
MnCl2-4H20 1.6 mg/L 8.1 µM

Dissolve 5g EDTA in 800 mL water and adjust the pH to 7.5 with NaOH. Then add the other components in the quantities mentioned below and add water to a final volume of 1L. Sterilize the solution over a 0.22µm filter

498 mg FeCl3(anhydrous)
84 mg ZnCl2
765 µL 0.1 M CuCl2-2H20 1.70 g/100 mL
210 µL 0.2 M CoCl2-H2O 4.76 g/100 mL
1.6 mL 0.1 M H3BO3 0.62 g/100 mL
8.1 µL 1M MnCl2-4H20 19.8 g/100 mL

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

Adjust to 1L with distilled H2O. Sterilize by autoclaving.


[Buffers, Solutions]

Biotin 500x stock solution

- Dissolve 20 mg biotin in 100 mL of 0.05 M NaOH solution and filter sterilize

- Store at 4 °C

- Durable for one year


TAE buffer

For 1 L of 50 x TAE buffer you need:

- 242.48 g Tris

- 41.02 g Sodiumacetate

- 18.612 g EDTA

- Adjust pH to 7.8 with acetic acid

- Solve in dH2O

10 mL of the stock is diluted in 1 L dH2O for the gel electrophoresis (0.5 x TAE buffer)


Cold osmotic shock buffers for the release of periplasmic protein fraction

Cell fractionating buffer 1 (pH 8)

• 0.2 M Tris

• 200 g L -1 sucrose

• 0.1 M EDTA


Cell fractionating buffer 2.1 (pH 8)

• 0.01 M Tris

• 0.005 M MgSO4


Cell fractionating buffer 2.2 (pH 8)

• 0.01 M Tris

• 0.005 M MgSO4

• 1% Trition

• 2% SDS


Cell fractionating buffer 2.3 (pH 8)

• 0.01 M Tris

• 0.005 M MgSO4

• 1% Trition

• 0,2% SDS







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