Phenol Chloroform Extraction

Author:

Whitney Tsai, Jessie Salter

Copyright:

This documentation is available under a Creative Commons (`CC-BY`_) license.

Modification History

See Phenol Chloroform Extraction (for toepads) History

Purpose

Preparation

DTT

  1. Make a stock solution of 1M DTT (store in aliquots at -20˚C, avoid freeze/thaw cycles)

  2. The molecular weight of DTT is #.25g, so add #.5425 g DTT to 10 ml of ultra-purse ddH2O

Buffer ATL

  1. TODO: Needs to be completed

STE Buffer

  1. TODO: Needs to be completed

Toepad Collection

  1. Cut toe pad with a clean scalpel into a piece of curled foil

  2. Place toe pad in #.5 mL tube

  3. Change foil and scalpel blade and flame sterilize forceps between each sample

  4. Can stop at this step and store toe pads dry in the freezer

Steps

Day 1

Toe Pad Wash (for large chunks; can skip small/flaky samples)

  1. Preheat Buffer ATL on heat block to dissolve precipitate

  2. Add 500 ul 100% ethanol to each tube

  3. Incubate samples on a thermomixer at room temperature at 1000 RPM for 5 minutes

  4. Remove ethanol and discard

  5. Add 500 ul 100% ethanol to each tube

  6. Incubate samples on a thermomixer at room temperature at 1000 RPM for 5 minutes

  7. Add 500ul of 1X STE Buffer to each tube

  8. Incubate on a thermomixer at room temperature at 1000rpm for 3-4 hours

  9. Remove STE Buffer and discard

  10. Add 500ul of 1X STE Buffer to each tube

  11. Incubate on a thermomixer at room temperature at 1000rpm for 3-4 hours

  12. Remove STE Buffer and discard

  13. Proceed to Toe Pad Digestion

Toe Pad Digestion

  1. Remove STE Buffer and discard; add the following to each tube containing a toe pad:

    1. 180 ul Buffer ATL

    2. 20 ul Proteinase K

  2. For large chunks, use flame-sterilized forceps to break up toepad as much as possible

  3. Vortex and place in thermomixer at 56˚C at 1000 rpm for ~2 hours

  4. Remove from thermomixer

  5. Using a separate mini pestle for each sample, mash tissue in tubes (REPEAT this step every few hours if necessary – we try and avoid using mini pestles if possible, so as not to lose material)

  6. Vortex and return to thermomixer and incubate at 56˚C at 1000 rpm overnight

Day 2

Toe Pad Digestion

  1. Remove from thermomixer and add 25 ul 1M DTT to each sample

  2. Vortex, return to thermomixer, and incubate at 56˚C at 1000 rpm for at least 1 hour

  3. Remove from thermomixer and add 15ul proteinase K

  4. Vortex well and place in thermomixer at 56˚C at 1000 rpm for 30 minutes

  5. If tissue is completely digested, move to Step X. If tissue not digested, continue incubating and smush with mini pestle every few hours until sample is completely digested

Phenol-Chloroform addition

  1. Spin down Phase Lock Gel Light tubes at 12,000 RPM for 30 seconds

  2. Vortex sample after removing from incubation. Spin down quickly.

  3. Transfer sample to pre-spun Phase Lock Gel tube

  4. In fume hood, add 225ul Phenol:Chloroform:Isoamyl Alcohol (24:25:1)

  5. In fume hood, mix thoroughly by manually rotating tube for 10 minutes

  6. In fume hood, open lid to each tube to vent gas that has built up in each tube

  7. In fume hood, centrifuge at 14,000 RPM for 15 minutes

  8. While tubes are spinning, label a batch of 1.5 mL tubes for final storage (include initial tube number)

  9. In fume hood, pipet the supernatant to final storage tubes while being careful not to disturb the interface between the two layers. It may be easiest to pour the supernatant into the final tubes rather than pipetting (if you do puncture the interface, return all liquid to the phase lock tube and repeat step 7). Dispose of Phase Lock Gel Light tubes appropriately.

  10. Add 20 ul 3M NaOAc to each final storage tube

  11. Add 500 ul cold 100% ethanol

  12. Mix well and store in -20˚C for at least 30 minutes. Tubes can remain at -20˚C overnight, which may increase DNA yields while also, possibly, increasing impurities in the final extract.

Precipitation

  1. Remove tubes from -20˚C, and centrifuge at 14,000 RPM for 10 minutes

  2. Pour off supernatant, being careful not to dislodge the DNA pellet

  3. Add 500ul cold 70% ethanol to each sample without disturbing the DNA pellet

  4. Centrifuge at 14,000rpm for 10 minutes

  5. Pour off supernatant and discard, being careful not to dislodge the DNA pellet

  6. Leave the tubes open and dry the DNA pellet in the fume hood (2-3 hours)

  7. Add 50ul 10mM Tris-HCl pH 7.5 to each tube and close the tube.

  8. Store tubes at 4˚C for 24 hrs or bench top overnight

  9. Proceed to DNA quantification