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RNA, DNA and Protein Isolation from ES Cells

RNA

  1. From a single 3-day confluent well of a 6-well dish, scrape cells off bottom and pellet.
  2. Add 0.5 ml TRIzol® Reagent, let sit at RT for 10 min and follow rest of procedure from TRIzol protocol, with volumes cut in half (since protocol starts with 1 ml TRIzol Reagent).
  3. Resuspend final pellet in 20 µl formamide and store at -80°C.
  4. Determine RNA concentration by spectrophotometry, making sure the blank cuvette has 1 µl formamide in it.

    **Always run sample of RNA on agarose gel to check for degradation.**

DNA

  1. From a single 3-day confluent well of a 6-well dish, scrape cells off bottom and pellet.
  2. Add 0.5 ml ES cell DNA digest buffer* and digest O/N at 55-60°C.
  3. Precipitate DNA by adding 1 ml of [ice-cold ethanol + 15 µl 5M NaCl]. Keep on ice for 15 minutes.
  4. Spin down 4°C for 15 minutes.
  5. Wash pellet with 70% ethanol.
  6. Resuspend in 50-100 µl H2O.
  7. Determine concentration by spectrophotometry.

*ES Cell DNA Digest Buffer

  • 10mM Tris, pH 7.5
  • 10mM EDTA
  • 10mM NaCl
  • 0.5% Sarkosyl
  • 1 mg/ml Proteinase K

Protein

  1. From a single 3-day confluent well of a 6-well dish, scrape cells off bottom and pellet.
  2. Add 0.25 ml RIPA with protease inhibitors and, if you are looking at histones, Na butyrate added.
  3. Let lyse on ice for 20 minutes.
  4. Sonicate for 10 pulses at 30% power.
  5. Spin out debris for 10 minutes at 4°C.
  6. Transfer aqueous to new tube. You can store at -20°C.
  7. Use Bradford assay to determine concentration of total protein.

To Precipitate Protein

  1. Add 8 volumes acetone: 2 volumes protein solution.
  2. Place on ice for 30 minutes.
  3. Spin for 10 minutes, 4°C, at maximum speed.
  4. Discard supernatant.
  5. Use SpeedVac to finish drying pellet.
  6. Resuspend in RIPA + loading dye.

Solutions

RIPA

  • 1x PBS
  • 1% Nonidet® P40
  • 0.5% Sodium Deoxycholate
  • 0.1% SDS

500 ml

  • 50 ml 10x PBS
  • 5 ml
  • 2.5 g
  • 5 ml 10%

Protease Inhibitors

  • 10 µg/ml PMSF
  • 1 µM Pepstatin
  • 1 µM Leupeptin

© 2014 The University of Texas MD Anderson Cancer Center