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Transformation and Plating
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(New page: ==Transformation Protocol== Before you start, make sure the water bath is at 42 degrees and the SOC is at 37 degrees. If the SOC is cloudy or has anything floating in it, it has gone bad...)
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Revision as of 17:32, 28 October 2008
Transformation Protocol
Before you start, make sure the water bath is at 42 degrees and the SOC is at 37 degrees. If the SOC is cloudy or has anything floating in it, it has gone bad and you'll have to make more. Also, make sure you have enough plates and check the resistance of the DNA before plating.
1. All bacterial cells are found in the -80 in the top section. For ligations, use ultracompotent cells. For all other transformations use excel10 cells. Thaw the cells and the DNA on ice for 7-8 min.
2. Take 25 ul of the cells and place them in a different 2ml microcentrifuge tube. Add 1ul of DNA for regular transformations or 3ul-5ul of DNA for ligation transformations. Let the tubes sit on ice for 30 min.
3. Place the tubes in the water bath for exactly 30 sec, and then place them on ice for 2min.
4. Put 900ul SOC in each tube and place in the 37 degree shaker for 60 min.
5. Take tubes out of the shaker and centrifuge at 6,000rpm for 1min. You should see a white pellet at the bottom.
6. Remove .85ml from the each tube and discard. Resuspend the remaining 50ul.
7. Plate the 50ul. First, place the transformed cells on the plates. Then spread the cells across the entire plate.
8. Place the plates upright in the incubator for 20 min. After this, flip the plates and leave them in the incubator for 12-14 hrs.
