Peqlab RNA/DNA/protein precipitation protocol
RNA Isolation
1
For tissue samples: Homogenize the tissue in 1mL TriFast reagent.
For cells grown in monolayer: Lyse the cells directly in the culture dish by addition of 1mL
TriFast reagent and passing the cell lysate several times through a pipette. (The amount of
TriFast reagent required is proportional to the area of the well (1ml/10cm^2); and not on cell
density).
TIP: Insufficient amount of reagent results in contamination of RNA with DNA.
2
Keep samples at RT for 5 min
Add 0.2 ml of pure chloroform per 1ml of TriFast reagent added (without isoamyl alcohol)
Shake vigorously (DO NOT vortex) for 15 seconds
Keep at RT for 5 minutes.
Spin at 12,000g for 5 min at 4*C
3
Take the the aqueous phase (colorless upper phase) for RNA – Save the lower phase for DNA
and the interphase for protein at 4*C.
Transfer the aqueous phase to a fresh tube. Add 0.5 ml of isopropanol per 1 ml of TriFast
added.
Keep samples on ice for 10 min
Spin at 12,000g for 10 min at 4*C
4
Remove the supernatant gently
Wash the RNA precipitate twice with 75% ethanol by vortexing and subsequent
centrifugation for 10 min at 12000g at 4*C
Air-dry the samples
Resuspend the pellet in RNAase-free water
Store at -20*C (The A260/280 ration should be 1.6-2.0)
DNA Isolation
1
Remove the phenol phase (from step 3)
Add 0.3 ml of 100% ethanol per ml of TriFast reagent added
Mix well by inversion (DO NOT vortex)
Keep at RT for 2-3 min
Spin at 2,000g at 4*C for 15 minutes
2
Remove the supernatant (Store at 4*C for protein extraction)
To the pellet, add 1 ml of 0.1M Na-citrate in 10% ethanol. Keep at RT for 30 min.
Spin at 2,000g at 4*C for 5 min. Repeat this citrate wash step again.
3
Suspend the DNA pellet in 2ml of 75% ethanol.
Keep at RT for 15 minutes with periodic mixing (NO vortexing).
Spin at 2,000g for 5 min at 4*C
4
Dry the pellet briefly for 10-15 min
Dissolve it in 8mM NaOH (about 200 uL) using a wide bore pipette.
Adjust the final DNA concentration between 0.2-0.3 ug/ul with 8mM NaOH
Protein Isolation
1
To the supernatant from step 2 of DNA isolation: add 1.5 ml isopropanol.
Keep at RT for 10 min
Spin at 2,000g for 10 min at 4*C
2
Remove supernatant gently
Add to the pellet: 2ml solution of 0.3M guanidium hydrochloride in 95% ethanol.
Keep at RT for 20 minutes
Spin at 7,500g for 5 min at 4*C (Repeat the guanidium hydrochloride washing step thrice)
3
Add 5ml of 100% ethanol to the pellet
Keep at RT for 20 min
Spin at 7,500g for 5 min at 4*C
4 (Method for large amounts of starting material)
Remove ethanol by decantation and air-dry the pellet for 15 minutes
Dissolve it in 1% SDS using pipette. Incubation at 50-100*C may be required for complete
solubilization.
Insoluble material can be removed by centrifuging at 10,000g for 10 min at 4*C.
Transfer the supernatant to a fresh tube. This can be used immediately or stired at -20*C for
future use.
4’(Method for small amounts of starting material)
1. To extract proteins perform TRIzol® isolation according to the manufacturer's instructions.
2. Protein pellet was dried by centrifuging under vacuum for 10 min as suggested in the
TRIzol® protocol
3. Extract the vacuum-dried protein pellet with 2% (w/v) DEA in 50 mM NaCl, at 1:5 or 1:10
ratio (depending on the amount of original material used) for 20–60 min at room
temperature.
4. Centrifuge the extracts at 12,000°—g for 10 minutes at 4 °C.
5. Collect the supernatant and neutralize it with 20% volume of 0.5 M Tris–HCl, pH 6.8.
6. Keep at 4 °C for immediate use or at −20 °C for long-term storage.
J Biochem Biophys Methods. 2006 Aug 31;68(2):127-31
4” (Method for small amounts of starting material, quite long and expensive)
1) Load the phenol-ethanol supernatants into Spectra/Por® 6 regenerated cellulose (RC)
dialysis membranes (MWCO 2000; Spectrum Laboratories, Rancho Dominguez, CA, USA)
and dialyze it against three changes of an aqueous 0.1% sodium dodecyl sulfate (SDS)
solution at 4°C, changing the solution first after 16 h, then after 4 h, and again after 2 h,
respectively. (For every 1 mL phenol-ethanol supernatant, 100 mL 0.1% SDS solution are
used). During dialysis, the samples partitioned into three phases:
(i) a colorless supernatant (approximately 85% volume),
(ii) a globular mass (approximately 10% volume),
(iii) a colorless, viscous liquid (approximately 5% volume).
2) Concentrate the supernatant in the dialysis membrane for the purposes of protein content
determination by removing samples from the dialysis membrane and loade them into iCON™
Concentrators (20 mL capacity, 9K MWCO; Pierce) and centrifuged at 6000× g at room
temperature for 20 min in a swinging-bucket rotor to reduce the volumes from 12 mL to 100
μL.
3) Resuspend the globular mass, containing the bulk of the proteins, in 200 μL total solvent—
either 8 M urea in Tris-HCl, pH 8.0, 1% SDS in molecular biology-grade water or a 1:1
combination of the two.
BioTechniques 42:467-472 (April 2007)