Extraction of DNA from TRIZOL preparations

This protocol is for extracting DNA from TRIZOL preparations after the RNA-containing upper phase has been completely removed. It is based on the online protocol by Shirley Zhu of the Chang Lab (http://changlab.stanford.edu/DNAextractionfromTRIZOL_Organicphase_update).

Initial sample preparation

• Perform TRIZOL extraction as you normally would for RNA extraction, ie by mechanically disrupting tissues in Qialyser. Approximately 20 mg of tissue is sufficient.
• After removing the RNA-containing aqueous (upper) phase, re-centrifuge tubes (now containing only the interphase and organic (lower) phase) at 12,000 G for 5 min at 4 deg C.
• Remove any remaining upper phase, taking care to remove everything without damaging the interphase. Samples can now be stored at 4 deg C for days to a couple of weeks.

Materials

• Back Extraction Buffer: Containing 4 M Guanidine Thiocyanate (FW 118.16), 50 mM Sodium Citrate NaCl (FW 294.1) and 1 M Tris base (FW 121.14), prepared in ddH2O and sterile filtered. For 25 mL, combine 11.82 g Guanidine Thiocyanate, 0.37 g Sodium Citrate NaCl and 3.03 g Tris base. Be careful to add small volumes of water, gradually, as the reagents will come up to the required volume VERY quickly.
• Isopropanol
• 70% Ethanol
• Qiagen Elution Buffer (or similar TE buffer)

Extraction steps

1. Add 500 uL of Back Extraction Buffer (BEB) for every 1 mL of TRIZOL used in the initial extraction. Place on shaker for 10 min. Always mix by inversion or use the shaker. NEVER vortex samples, as it will destroy the DNA.
2. Centrifuge tubes at 12,000 G for 30 min at room temperature.
   1. Note: After this step is complete, set the centrifuge to cool to 4 deg C.
3. Transfer aqueous (upper) phase to a clean tube and add 400 uL of ice cold isopropanol (to precipitate the DNA). Mix by inversion. Incubate at room temperature for 5 min.
4. Centrifuge tubes at 12,000 G for 15 min at 4 deg C.
5. Very carefully remove supernatant (it is very unlikely that the DNA pellet will be visible). Add 500 uL of 70% ethanol and mix by inversion (to wash the pellet- this step removes the salt that co-precipitates with the DNA).
6. Centrifuge tubes at 12,000 G for 15 min at 4 deg C.
7. Very carefully remove all of the supernatant (once again taking care not to disturb what is probably an invisible DNA pellet).
8. Dissolve DNA pellet in 50 uL of Qiagen elution buffer. Samples can now be stored at 4 deg C.
   1. Note: Additional washing steps (using phenol, chloroform and isoamylalcohol) should be performed if a more pure pellet is required, however, this preparation should be sufficient for running qPCR/copy number experiments.