"

From 2012.igem.org

Home Homepage News Team Members Focus Groups Seminars Work Impressions Project Our Project Results Discussion/Outlook Lab Work Flow Notebook Overview Materials Methods Computational Data Bioinformatical Tools iGEM What is iGEM? Parts Submitted Attributions Safety > Göttingen City University of Göttingen Max-Planck-Institute S1-Demo Lab Human Practice Our Human Practice Projects Public and Media Survey Synthetic Biology Day Panel Discussion Flash Coli Sponsoring Our Sponsors Interested in...?
Deutsch  / English

Contents 1 Recipes 1.1 Media 1.1.1 LB-Medium 1.1.2 M9 Swimming Agar 1.1.3 Tryptone swimming agar 1.2 Antibiotics 1.3 Supplements 1.4 Buffer and Solution 1.4.1 5x M9 salts stock solution 1.4.2 CaCl2 Buffer for competent cells 1.4.3 50x TAE for agarose gels 2 E. coli 2.1 Strains 2.2 Storage 2.2.1 Glycerol Stock

Recipes

Media

Recipes for liquid and solid media for bacterial cultivation. Stir until dissolved; sterilize by autoclaving 121 °C. Do not forget a magnetic stirrer.

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

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

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.

Reagent	Abbreviation	Stock Conc. [mg/mL]	Working Conc. [µg/mL]	Dilution	Solvent	Notes
Ampicillin	Amp	100	100	1,000x	ddH2O	Culture plates with Amp can be stored at 4 °C for about 2 weeks. Stock solutions can be stored at 4 °C for 2 weeks but can last as long as 4-6 months when stored at -20 °C.
Chloramphenicol	Cm	20	20	1,000x	EtOH
Kanamycin	Kan	50	50	1,000x	ddH2O	Plates may only last a few weeks at 4 °C.

Supplements

• Aminoacid mix (containing all aminoacids except tryptophane): stocksolution 0.2 % (w/v), varying amounts used in swimming assays, view methods and notebook of group 1
• Caffeine: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
• D-Aspartate: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
• 2-Ethyl-1-Hexanole: stocksolution 5 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
• Geraniol: stocksolution 5 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
• L-Aspartate-4-Benzylester: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
• L-aspartate: stocksolution 10 mM, varying amounts used in swimming assays, view methods and notebook of group 1
• Leucin: stocksolution 30.49 mM, add to M9-agar: 4 mL (final concentration 0.305 µM)
• Methionin: stocksolution 26.8 mM, add to M9-agar: 4 mL (final concentration 0.268 µM)
• Sodium Cyclamate: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
• Tryptone: stocksolution 0.5 % (w/v), varying amounts used in swimming assays, view methods and notebook of group 1
• Vanillin: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods

Buffer and Solution

Recipes for stocks of buffers and solutions.

5x M9 salts stock solution

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

• Stir until dissolved

CaCl₂ Buffer for competent cells

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

• Stir until dissolved; sterilize by sterile filtration.

50x TAE for agarose gels

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

• Stir until dissolved. Working concentration is 1x (40 mL of 50x stock ad 2 L ddH₂O).

E. coli

Strains

E. coli strain list - List of the working E. coli strains including genotypes, genetic markers, and alleles.

Storage

Storage of E. coli strains - How to keep E. coli strains.

Glycerol Stock

The glycerol stock is prepared as follow:

• 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!

↑ Back to top!

sub>2