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August/3 August 2009
From 2009.igem.org
We transformed and amplified several parts from the registry distribution according to the following steps:-
1. The following parts were extracted from DNA distribution plates by dissolving with 15 ul MilliQ purified water: BBa_E0240 (GFP reporter device) BBa_E0430 (EYFP reporter device) BBa_E0420 (ECFP reporter device) BBa_J06702 (mCherry reporter device)
2. 5 ul extracted DNA was mixed with 100 ul thawed competent E. coli culture, then left on ice for 30 min.
3. The cultures were heat shocked at 42 degrees Celsius for 2 min, then immediately put on ice again for 2 min.
4. 900 ul LB nutrient solution was added, making a total of 1 ml of solution per DNA part.
5. Cultures were incubated at 37 degrees Celsius for 1 hr.
6. Meanwhile, culture dishes (containing LB culture medium + agar) were taken out from cold storage and dried in low-temperature oven.
7. Incubated cultures were ultracentrifuged at 14000 rpm for 2 mins to precipitate E. coli, then excess supernatant discarded, leaving about 150 ul of cell culture each.
8. Each cell culture was pipetted onto beads spread over the base of a culture dish, then smeared over the dish surface by briskly shaking the covered dish.
9. The parts were then put into cold storage.
In addition, we prepared some extra culture dishes for future use according to the laboratory standard protocols. For 500 ml of culture broth: 500 ml Milli-Q purified water 5.0g Bacto tryptone 2.5g Bacto yeast extract 2.5g sodium chloride 7.5g agar powder 0.5 ml Amphicillin or 2.5 ml Kanamycin solution for antibiotic resistance selection
Culture broth ingredients (apart from antibiotic solution) were mixed with a magnetic mixer, autoclaved and left to cool until the temperature was low enough to be touched with bare hands. Then antibiotic solution was added, and the complete culture broth was poured into culture dishes inside a 'Clean Bench'.
Points to remember: 1. Spray hands and workplace with (70%) ethanol to sterilize before beginning work. 2. Tilt the triangular flask containing culture broth to about 45 degrees, sear the mouth of the flask with Bunsen burner and keeping the flask tilted at an angle, pour broth into about 4-5 petri dishes consecutively. 3. Avoid bubble formation. Pour cleanly but quickly. 4. Turn off light, fan, and GAS after use!
