Calgary/protocols
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Revision as of 18:28, 12 October 2007
- Thaw 100 ul of competen cells (per transformation) on ice just before they are needed
- Add DNA (max 20ul) thawed cells and mix by flicking the side of the tube. Leave on ice for 30 minutes
- Heat shock for 2 minutes at 42 degrees Celsius or 5 minutes at 37 degrees Celsius
- Place on Ice for 5 minutes
- Add 250ul SOC medium to each tube
- Incubate for 30 to 60 minutes with shaking at 37 degrees Celsius. (Note that for Kanamycin containing plasmides always use one hour)
- Spin down to remove all supernatant except approximately 100 ul
- Plate approximately 30 ul on each of two antibiotic plates
- Grow overnight at 37 degrees Celsius
For this protocol we used three controls
- Positive Control - pBluescript in TOP10 cells on amp resistant plates
- Negative Control - TOP10 cells grown on amp resistant plates
- Negative Control - DB31 cells on amp resistant plates
Biobrick parts are shipped from the registry in a dehydrated from. As such they must be rehydrated before they can be used.
- Puncture a hole through the foil with a pipette tip into the well that corresponds to the Biobrick - standard part that you want
- Add 15 ul of diH20 (deionized water)
- Take 1 ul DNA and transform into your desired competent cells, plate out onto a plate with the correct antibiotic and grow overnight. Your goal here is to obtain single colonies
For this protocol we used three controls
- Positive Control - pBluescript in TOP10 cells on amp resistant plates
- Negative Control - TOP10 cells grown on amp resistant plates
- Negative Control - DB31 cells on amp resistant plates
