Team:NTNU Trondheim/Experiments

Protocols

Throughout the project we used and developed several protocols for creation of culture media and conduction of experimental procedures. Among these were phage isolation, creation of supercompetent cells, transformation, DNA purification, restriction digestion, gel electrophoresis, TECAN analysis and PCR. The procedures where conducted as described below unless otherwise described in the lab journal.

Media

All media should be mixed using a magnetic stirrer in a bottle twice as large as the liquid volume, and autoclaved at 121 °C for 20 minutes.


Luria-Bertani broth (LB)
· Reverse osmosis water (1 L)
· Bacto tryptone (10 g)
· Yeast extract (5 g)
· NaCl (10 g)


Luria-Bertani agar (LA)
· LB medium (1 L)
· Agar (15 g)


Phosphate-buffered saline (PBS)
· Distilled water (1 L)
· NaCl (8 g)
· KCl (0.2 g)
· Na2HPO4 (1.42 g)
· KH2PO4 (0.24 g)
 

NB: If you are making agar plates, add 15 g agar to the solution before autoclaving. Add the appropriate antibiotic to the agar solution after cooling the solution to 50 °C.

Antibiotics

Work in a fume hood when preparing antibiotic stock solutions. For each solution, dissolve the antibiotic in water or ethanol, and sterile filter through a 0.2 μm filter. Aliquot into 1.5 mL Eppendorf tubes and store in the freezer at -20 °C.


Kanamycin (50 mg/mL stock solution)
· Kanamycin (250 mg)
· Reverse osmosis water (5 mL)


Ampicillin (50 mg/mL stock solution)
· Ampicillin (250 g)
· Reverse osmosis water (5 mL)


Chloramphenicol (35 mg/mL stock solution)
· Chloramphenicol (175 mg)
· 96% ethanol (5 mL)


Spectinomycin (100 mg/mL stock solution)
· Spectinomycin (500 mg)
· Reverse osmosis water (5 mL)

Experiments

Throughout the project we used and developed several protocols for experimental procedures. Among these were phage isolation, creation of supercompetent cells, transformation, DNA purification, restriction digestion, gel electrophoresis, TECAN analysis and PCR. The procedures where conducted as described below unless otherwise described in the lab journal.