Team:SDU-Denmark/Protocols/Transformation

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(New page: 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...)
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Protocol for transforming (E.coli)
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=Protocol for transforming (E.coli)=
   
   

Revision as of 15:47, 24 July 2009

Protocol for transforming (E.coli)

  1. 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 (controle of the cells) and 1 tube with puc plasmid which is supercoiled and known to work (controle of our plasmid).
  2. Store on ice for 40 min.
  3. Keep at 42º C for 2 min.
  4. Keep on ice for 5 min.
  5. Add 1 ml LB to each tube.
  6. The tubes are stored at 37 º C for 2 hours while being shaken.
  7. Centrifuge the tubes at 3500 rounds pr. minute for 5 min.
  8. Suck up and throw out 850 µl and resuspend the remaining by pipetting up and down.
  9. 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.
  10. Store at 37 º C over night.
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