Sean Lauber:DNA Extraction Using Trizol

From OpenWetWare

Jump to: navigation, search

Procedure

  1. Remove media on cells and add 1 ml of Trizol (regardless of plate size). DO THIS ON THE BENCH AND NOT IN THE TISSUE CULTURE HOOD
  2. Scrape cells and pipette into a 1.5 ml eppendorf
  3. Pipette up and down to lyse cells
  4. Incubate at RT for 5 min. At this point you can store the sample at -80*C or continue
  5. In a fume hood, add 200 μL chloroform, shake to mix
  6. Incubate at RT for 3 min
  7. Spin at 13K RPM/4*C/15 min
  8. Pipette 350 μL of the top (aqueous) layer into a new tube (store at -80*C) or discard. This is the RNA-containing layer, if you don't want the RNA, discard this.
  9. Add 300 μL of 100% EtOH to the organic/interphase layers (what's left in the tube after removing the top layer), vortex
  10. Incubate at RT for 3 min
  11. Spin 12K RPM/RT/5 min
  12. Discard supernatant
  13. Wash pellet twice with 1 ml 0.1 M Na Citrate x 2
  14. Resuspend pellet in 1 ml 75% EtOH
  15. Incubate at RT for 15 min, vortexing periodically
  16. Spin 12K RPM/RT/5 min
  17. Discard supernatant and dry at 55*C
  18. Dissolve pellet in 50 μL sterile/nuclease-free water at 55*C for 10 min. OPTIONAL STEP: spin at 12 K RPM/4*C/10 min to pellet the insoluble material and transfer the supernatant (containing the DNA) to a new tube
  19. Store at -20*C

Solutions required

0.1 M Na Citrate

  For 10 ml of 0.1 M:
  *Dissolve 0.29 g into 9 ml water
  *Add 1 ml of 100% EtOH
Personal tools