Sean Lauber:DNA Extraction Using Trizol
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Procedure
- 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
- Scrape cells and pipette into a 1.5 ml eppendorf
- Pipette up and down to lyse cells
- Incubate at RT for 5 min. At this point you can store the sample at -80*C or continue
- In a fume hood, add 200 μL chloroform, shake to mix
- Incubate at RT for 3 min
- Spin at 13K RPM/4*C/15 min
- 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.
- Add 300 μL of 100% EtOH to the organic/interphase layers (what's left in the tube after removing the top layer), vortex
- Incubate at RT for 3 min
- Spin 12K RPM/RT/5 min
- Discard supernatant
- Wash pellet twice with 1 ml 0.1 M Na Citrate x 2
- Resuspend pellet in 1 ml 75% EtOH
- Incubate at RT for 15 min, vortexing periodically
- Spin 12K RPM/RT/5 min
- Discard supernatant and dry at 55*C
- 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
- 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