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Team:Goettingen/Project/Materials
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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.
| 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
- 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
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
BL21DE3
Genotype
- E. coli B F- dcm ompT hsdS(rB- mB-) gal [malB+]K-12(λS)
Genetical Characteristics
- Antibiotic resistance: none!
Reference
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
MG1655
Genotype
- F-λ-, rph-1
Genetical Characteristics
- Antibiotic resistance: none!
Reference
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
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
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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