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Team:Evry/Protocols/04
From 2013.igem.org
Solutions, Media and Petri Dishes
Antibiotics
Carbenicillin:
Put 150 mg of Carbenicilline in 15 mL of distilled water (10 mg per mL).
Filter with a 0,22 µm millipore,divide in 2 mL tubes and store at -4 °C.
Use 1 µL of Carbenicillin for 1 mL of solution.
Chloramphenicol:
Put 525 mg of Kanamycin in 15 mL of ethanol (35 mg per mL).
Filter with a 0,22 µm millipore,divide in 2 mL tubes and store at -4 °C.
Use 25 µL of Chloramphenicol for 1 mL of solution.
Kanamycin:
Put 750 mg of Kanamycin in 15 mL of distilled water (50 mg per mL).
Filter with a 0,22 µm millipore,divide in 2 mL tubes and store at -4 °C.
Use 1 µL of Kanamycin for 1 mL of solution.
Spectinomycin:
Put mg of Spectinomycin in 15 mL of ( mg per mL).
Filter with a 0,22 µm millipore,divide in 2 mL tubes and store at °C.
Use µL of Spectinomycin for 1 mL of solution.
Thoses antibiotics are used to select bacteria with a plasmid including an interest construction and the corresponding antibiotic resistance gene.
LB and LB Agar
Mix 20 g of LB Broth in 1L of distilled water in a bottle of 1L, then put in autoclave.
Mix 17,5 g of LB Agar in 500 mL of distilled water in a bottle of 1L, then put in autoclave.
M9 Medium
Composition for 50 mL of:
| Reagent | M9 medium (without iron) | M9 medium (with iron) |
|---|---|---|
| M9 salt (5X) | 10 mL | |
| CaCl2 (1M) | 5 µL | |
| MgSO4 (1M) | 100 µL | |
| Glycerol (50%) | 800 µL | |
| Thiamine | 5 µL | |
| NaOH (pH 7.4) | 12.5 µL | |
| H2O | 40 mL | 39 mL |
| FeSO4 (10mM) | - | 50 µL |
| Casamino acids (0.2%) | - | 1 mL |
Once the mixture is prepared, the medium must be filtered to be sterilised using 0.22 µm filter.
Petri dish
Melt the LB Agar in a microwave, and split it in 50 mL tubes.
When the tubes have cooled, put the necessary amount of antibiotics in it (50 µL for Carbenicillin and Kanamycin, 1250 µL for Chloramphenicol) and mix thoroughly.
Put 25 mL of the solution in a Petri dish.
CaCl2 and CaCl2+glycerol
Solution for 1M Cacl2:
Add 14,30g of CaCl2 into 100 ml desalted water
Solution for 0,1M Cacl2:
Add 50 mL of CaCl2 1M solution into 450 ml of desalted water
Solution for 0,1M Cacl2 + 15% glycerol:
Add 50 mL of CaCl2 1M solution and 75 mL of glycerol 100% into 450 ml of desalted water
Tris-Acetate-EDTA (TAE) Buffer solutions
NaOH solution 5M 100 mL:
Add 19,99 g of NaOH in 100 mL of distilled water.
EthyleneDiamineTetraacetic Acid (EDTA) solution 0,5M 300 mL:
Add 43,8 g of EDTA in 250 mL of distilled water.
Add NaOH 5M until EDTA is solubilized and pH~8.
Add distilled water until you reach 300 mL.
TAE Stock solution 50X 300 mL:
Add 60,5 g of TAE in 187,5 mL of distilled water.
Add 14,27 mL of acetate solution (previously put at 4°C).
Add 25 mL of EDTA solution previously prepared.
Add distilled water until you reach 250 mL.
TAE solution 1X 100 mL:
Add 5 mL of TAE Stock solution 50X in 100 mL of distilled water.
Tris HCl
Disolve 4,6g of Trisbase in 30 mL of distilled water.
Adjust pH with concentrated HCl(~4 mL) until pH=7,5.
Add distilled water until 50 mL then put in autoclave.
If the solution has a yellow coloration, do the preparation again with better Trisbase.
Tris HCl is used to store DNA.
