Sean Lauber:DNA Extraction Using Trizol

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

Solutions required

Na Citrate

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