IGEM:PennState/2007/Dnaprep

From OpenWetWare

< IGEM:PennState | 2007
Revision as of 15:10, 22 June 2007 by Luw134 (Talk | contribs)
Jump to: navigation, search

Telt Method -DNA Plasmid Preperation

note: This method is "not very good" in that it does not seperate plasmid DNA from chromosomal DNA. The advantage of this is that if there is no plasmid, you will get the chromosomal DNA. Use this prep to seperate the chromosomal DNA when there is no plasmid in the cell.

  • Use sterile techniques throughout
  • all centrifuge steps are at room temperature
  • put a tube of 100% ethanol on ice at start of procedure
  1. Grow cells overnight in 3mL LB (with appropriate antibiotics and sugars) in a 13 x 100mm tube at 37C (or other appropriate temperature) in a rotator.
  2. Pour 1.5 mL into sterile microcentrifuge tube. Centrifuge cells for 30 sec. Remove supernatent by aspiration
  3. Resuspend cells in 100uL TELT buffer. Vortex to resuspend pellet.
  4. Add 100uL 1:1 Phenol/Chloroform.
  5. Vortex for 15 sec. (Can leave cells for 5 minutes on th ebench).
  6. Centfigue for 1min. Transfer top layer to clean tube.
  7. Add 100uL chloroform/isoamyl (24:1)
  8. Vortex for 15 sec. (Can leave cells for 5 minutes on th ebench.)
  9. Centrifuge for 1 min.
  10. Transfer 75uL of the supernatent to a new microcenttrifuge tube.
  11. Add 200uL of 100% EtOH (cold). Mix well by inversion. Incubate at room temperature 2-10 minutes.
  12. Centifuge for 10 minutes.
  13. Pour off supernatant. Add 1mL 70% EtOH. Invert several times. Pour off liquid. Spin briefly to bring liquid to bottom of tube. Remove rest of liquid by aspiration, being careful not to disturb pellet.
  14. Air dry pellet (about 1/2 hour). or vacuum dry 5 minutes.
  15. Resuspend pellet in 30uL TE buffer.
  • Use caution when using phenol. If you get any on you wash immediately with lots of water, it will turn your skin white. Eyeprotection is suggested.
Personal tools