Team:Goettingen/Project/Materials

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Recipes

Media

Recipes for liquid and solid media for bacterial cultivation.

1. LB-Medium
1% (w/v) bacto tryptone
0.5% (w/v) bacto yeast extract
1% (w/v) NaCl
if required: ad 1% (w/v) agar


2. M9 Swimming Agar
1.25% (v/v) glycerol
20% (v/v) 5x M9 salt stock solution
0.1% (v/v) of CaCl₂ - 2H₂O stock solution (20 mg/mL)
0.1% (v/v) MgSO₄ stock solution (0.12 g/mL)
0.3 % (w/v) of agar


3. Tryptone swimming agar
1% (w/v) bacto tryptone
0.5% (w/v) NaCl
0.3% (w/v) agar

Antibiotics

Stock solutions and concentrations of antibiotics.

<h3 style="margin:3px; background:#cedff2; font-size:120%; font-weight:bold; border:1px solid #a3b0bf; text-align:left; color:#000; padding:0.2em 0.4em;"<a name="Supplements"></a>Supplements</h3>
Stock solutions and concentrations of supplements.

1. Aminoacid mix (containing all aminoacids except tryptophane): stocksolution 0.2% (w/v)
varying amounts used in swimming assays, view <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a> and notebook of group 1
2. Caffeine: stocksolution 10 mM
100 µl applied to whatmanpaper in swimming assays, view <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>
3. D-Aspartate: stocksolution 10 mM
4. 2-Ethyl-1-Hexanole: stocksolution 5 mM
100 µl applied to whatmanpaper in swimming assays, view <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>
5. Geraniol: stocksolution 5 mM
100 µl applied to whatmanpaper in swimming assays, view <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>
6. L-Aspartate-4-Benzyl: stocksolution 10 mM
100 µl applied to whatmanpaper in swimming assays, view <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>
7. L-aspartate: stocksolution 10 mM
varying amounts used in swimming assays, view <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a> and notebook of group 1
8. Leucin: stocksolution 30.49 mM
add to M9-agar: 4 ml (final concentration 0.305 µM)
9. Methionin: stocksolution 26.8 mM
add to M9-agar: 4 ml (final concentration 0.268 µM)
10. Sodium Cyclamate: stocksolution 10 mM
100 µl applied to whatmanpaper in swimming assays, view <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>
11. Tryptone: stocksolution 0.5% (w/v)
varying amounts used in swimming assays, view <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a> and notebook of group 1
12. Vanillin: stocksolution 10 mM
100 µl applied to whatmanpaper in swimming assays, view <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>

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Recipes for stocks of buffers and solutions.

1. 5x M9 salts stock solution

Stir until dissolved; sterilize by autoclaving at 121°C.


Amount	Substance
64 g	Na2HPO4 × 7 H2O
15 g	KH2PO4
2.5 g	NaCl
5.0 g	NH4Cl
ad 1000 ml	ddH2O








2. CaCl₂ Buffer for competent cells

Stir until dissolved; sterilize by sterile filtration.


Amount	Substance	Notes
7.351 g	CaCl2 × 7 H2O	Alternative 0.1 M CaCl2.
15%	Glycerol
ad 500 ml	ddH2O





3. 50x TAE for agarose gels

Stir until dissolved. Working concentration is 1x (40 ml of 50x stock ad 2 l ddH₂O).


Amount	Substance
242 g	Tris base
57.1 ml	Glacial acetic acid
18.6 g	EDTA
ad 1000 ml	ddH2O

<a name="E._coli"></a>E. coli

<h3 style="margin:3px; background:#cedff2; font-size:120%; font-weight:bold; border:1px solid #a3b0bf; text-align:left; color:#000; padding:0.2em 0.4em;"<a name="Strains"></a>Strains</h3>
E. coli strain list - List of the working E. coli strains including genotypes, genetic markers, and alleles.

<h3 style="margin:3px; background:#cedff2; font-size:120%; font-weight:bold; border:1px solid #a3b0bf; text-align:left; color:#000; padding:0.2em 0.4em;"<a name="Strains_Storage"></a>Storage</h3>
Storage of E. coli strains - How to keep E. coli strains. </li>

1. </h3>Glycerol Stock
• 900 µl over night culture in LB-liquid medium
• 900 µl 50% sterile glycerol

Glycerol is extremely viscous. Hence, use cut off tips to pipette. Glycerol stocks were vortexed to ensure uniformly mixture and shock frozen in N₂ or dry ice. Then, cultures were stored at -80°C. Do NOT forget to fill in the according GMO S1 construction sheet!

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