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Team:SDU-Denmark/Protocols/Transformation
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| + | =Protocol for transforming (E.coli)= | ||
# Take 2 and 5 µl of the plasmid from the distribution plates and add it to 2 different tubes with 50 µl competent cells in each. As controls use 1 tube with competent cells only (control of the cells) and 1 tube with puc plasmid which is supercoiled and known to work (control of our plasmid). | # Take 2 and 5 µl of the plasmid from the distribution plates and add it to 2 different tubes with 50 µl competent cells in each. As controls use 1 tube with competent cells only (control of the cells) and 1 tube with puc plasmid which is supercoiled and known to work (control of our plasmid). | ||
Latest revision as of 10:05, 17 August 2009
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Protocol for transforming (E.coli)
- Take 2 and 5 µl of the plasmid from the distribution plates and add it to 2 different tubes with 50 µl competent cells in each. As controls use 1 tube with competent cells only (control of the cells) and 1 tube with puc plasmid which is supercoiled and known to work (control of our plasmid).
- Store on ice for 40 min.
- Keep at 42º C for 2 min.
- Keep on ice for 5 min.
- Add 1 ml LB to each tube.
- The tubes are stored at 37 º C for 2 hours while being shaken.
- Centrifuge the tubes at 3500 rounds pr. minute for 5 min.
- Suck up and throw out 850 µl and resuspend the remaining by pipetting up and down.
- Spread out 75 µl on 2 LA (LB + agarose) plates with antibiotics (100 µg/ml ampicillin). The competent cells of the controle are spread out on 1 LA plate with antibiotics and 1 without, since they are not resistant.
- Store at 37 º C over night.
