Edinburgh iGEM 2018

General Protocols

Agarose Gel

Weigh 1 g of agarose
Add 100 mL of TAE 1X
Heat up until the solution is homogeneous, avoiding boiling. If it boils, move away from the heat until it “calms down” and put it back on the heat until the agarose is completely dissolved
While heating, prepare the bed in which the gel will polymerize. Make sure that it is well balanced and tight
When homogeneous, add 10 µL of SYBR SAFE DNA Gel Stain to the solution and mix well
Pour the solution into the bed and clear all its bubbles with a pipette tip. Add in the comb and make sure it is secure
Mix the samples with loading dye in a 5:1 ratio. Put the samples into the wells, as well as 5 µL of DNA ladder into the first well

10 g tryptone
10 g NaCl
5 g yeast extract
Water
For selective medium, supplement with antibiotic as appropriate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin)

15 g agar agar
10 g tryptone
10 g NaCl
5 g yeast extract
Water
For selective medium, supplement with antibiotic as appropriate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin)

Inoculate a single colony of appropriate cells into 10ml LB. Add antibiotic if needed and culture o/n at 37°C, 220rpm
Inoculate 100ml LB with 1 ml o/n culture
Incubate at 37 °C, 220rpm until OD600 = 0.3-0.6 (approx. 2 hrs)
Transfer to 2 x 50 ml Falcon and leave on ice for 30 mins
Centrifuge at 400 x g, 5 mins, 4 °C
Resuspend pellet GENTLY in 25 ml ice cold 0.1 M MgCl2
Incubate on ice for 30 min
Centrifuge at 4000 xg, 5 min, 4 °C
Resuspend pellet GENTLY in 25 ml ice cold 0.1 M CaCl2
Incubate on ice for 30 min
Centrifuge at 4000 xg, 5 min, 4 °C
Resuspend pellet GENTLY in 1.25 ml ice cold CaCl2/Glycerol solution (1.7 ml 0.1 M CaCl2, 0.3 ml 100 % glycerol)
Aliquot 100ul and flash freeze on dry ice. Store at – 80 °C

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