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 2060 min at room
temperature.
4. Centrifuge the extracts at 12,000g for 10 minutes at 4 C.
5. Collect the supernatant and neutralize it with 20% volume of 0.5 M TrisHCl, 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 4C, 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
L.
3) Resuspend the globular mass, containing the bulk of the proteins, in 200 L 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)