Team:Goettingen/Project/Materials

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Latest revision as of 16:15, 26 September 2012

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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-broth

• 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 x 2 H₂O 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.

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

• Stir until dissolved

CaCl₂ Buffer for competent cells

• Stir until dissolved; sterilize by sterile filtration.

50x TAE for agarose gels

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

E. coli

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

Non-mutated strains

These strains may not contain any plasmids or resistances, because they should be used for transformation or other experiments.

DH10B

Genotype

• F- endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) λ-

Genetical Characteristics

• Antibiotic resistance: none

Reference

• Invitrogen

DH5α

Genotype

• F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–

Genetical Characteristics

• Antibiotic resistance: none

Reference

• Invitrogen

BL21DE3

Genotype

• E. coli B F- dcm ompT hsdS(rB- mB-) gal [malB+]K-12(λS)

Genetical Characteristics

• Antibiotic resistance: none!

Reference

• Merck Millipore

XL1 blue

Genotype

• endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F'[ ::Tn10 proAB+ lacIq Δ(lacZ)M15] hsdR17(rK- mK+)

Genetical Characteristics

• Antibiotic resistance: none!

Reference

• Merck Millipore

MG1655

Genotype

• F-λ-, rph-1

Genetical Characteristics

• Antibiotic resistance: none!

Reference

• Coli Genetic Stock Center

RP437

Genotype

• F-, thr-1, araC14, leuB6(Am), fhuA31, lacY1, tsx-78, λ-, eda-50, hisG4(Oc), rfbC1?, rpsL136(strR), xylA5, mtl-1, metF159(Am), thiE1

Genetical Characteristics

• Antibiotic resistance: none!

Reference

• Coli Genetic Stock Center

Mutated strains

∆tar

Genotype

• F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, Δtar-739::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514

Genetical Characteristics

• Antibiotic resistance: Kanamycin

Reference

• Coli Genetic Stock Center

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!

LB-agar

LB-agar plates were prepared as follow:

• 1% agar (w/v) was added to 1 L LB-broth
• LB-agar was autoclaved
• LB-agar was poured in plates

The E. coli strains were plated out and stored at 4°C. The plates were renewed every two weeks.

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