RNA, DNA and Protein Isolation from ES Cells
RNA
- From a single 3-day confluent well of a 6-well dish, scrape cells off bottom and pellet.
- 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).
- Resuspend final pellet in 20 µl formamide and store at -80°C.
- 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
- From a single 3-day confluent well of a 6-well dish, scrape cells off bottom and pellet.
- Add 0.5 ml ES cell DNA digest buffer* and digest O/N at 55-60°C.
- Precipitate DNA by adding 1 ml of [ice-cold ethanol + 15 µl 5M NaCl]. Keep on ice for 15 minutes.
- Spin down 4°C for 15 minutes.
- Wash pellet with 70% ethanol.
- Resuspend in 50-100 µl H2O.
- 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
- From a single 3-day confluent well of a 6-well dish, scrape cells off bottom and pellet.
- Add 0.25 ml RIPA with protease inhibitors and, if you are looking at histones, Na butyrate added.
- Let lyse on ice for 20 minutes.
- Sonicate for 10 pulses at 30% power.
- Spin out debris for 10 minutes at 4°C.
- Transfer aqueous to new tube. You can store at -20°C.
- Use Bradford assay to determine concentration of total protein.
To Precipitate Protein
- Add 8 volumes acetone: 2 volumes protein solution.
- Place on ice for 30 minutes.
- Spin for 10 minutes, 4°C, at maximum speed.
- Discard supernatant.
- Use SpeedVac to finish drying pellet.
- 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