# Dahlquist:Total RNA Purification from Yeast

This protocol is for the Ambion (LifeTechnologies) RiboPure Yeast Kit, catalog #AM1926, with specific modifications used in the Dahlquist Lab.

## Before You Begin

### When the kit is first opened

• Dispense 750 μL Zirconia Beads into each of the 1.5 mL screw cap tubes provided with the kit.
• Cut off the tip of the Zirconia bead bottle with a clean razor blade.
• Mark all of the screw cap tubes that come with the kit with a line ~2.5 cm from the bottom of the tube to estimate the volume.
• Pour the Zirconia Beads into all the tubes, distributing the beads so that all tubes are filled.
• Store the tubes in the -20C freezer.
• Add 40 mL of 100% ethanol to the Wash Solution 2/3 and check the box on the bottle.

### Immediately before beginning the procedure each time

• Clean the bench surface and pipettors with RNaseZap.
• Make sure that the water bath is set to 37°C and the heat block is set to 95°C.
• Check Wash Solution 1 and the 10% SDS for precipitation and redissolve by heating to 37°C if necessary.
• Get out and label all the tubes required in the procedure.
• Fill an ice bucket with ice.
• Set up your work station in the fume hood with racks, tips, waste container lined with a plastic back, and gloves.

## RNA Purification Procedure

### Initial RNA Purification

1. Thaw on ice the frozen cell pellets (in 50 mL conical tubes) from the Cold Shock and Recovery experiment that were stored at -80°C. Vortex to make sure that the cell pellets are completely thawed.
2. Resuspend cells in lysis reagents by adding the following in the order shown. Resuspend by vortexing vigorously for 10-15 seconds:
Amount     Component
480 μL     Lysis Buffer
48 μL     10% SDS
480 μL     Phenol:Chloroform:Isoamyl Alcohol

Note that the Lysis Buffer and 10% SDS can be added at the bench; the Phenol:Chloroform:Isoamyl Alcohol must be dispensed in the fume hood, as it is hazardous. All steps where Phenol:Chloroform:Isoamyl Alcohol is present must be performed in the hood.
1. Transfer the mixture of cells and lysis reagents to one of the prepared tubes containing the Zirconia Beads, and securely fasten the lid.
• Take care to get as much of the mixture as possible into the tubes.
• Because the tubes will get really full, some of the mixture may spill out. Be prepared to immediately wipe off the tube with a kim wipe and change your gloves if they become contaminated.
2. Beat cells for 10 minutes on the vortex mixer with the vortex adapter.
• Position the tubes horizontally, in a balanced configuration, on the vortex adapter with the tube caps towards the center. They will "click" when they are positioned correctly. Turn on the vortex mixer at maximum speed and beat for 10 minutes to lyse the yeast cells.
3. Spin the tubes for 5 minutes at room temperature at 10,000 rpm in the microcentrifuge. (Note that this is a departure from the manufacturer's protocol, which spins harder. I found that a harder spin will crack the tubes in the centrifuge and is unnecessary to separate the phases. Kam D. Dahlquist 19:22, 11 March 2016 (EST))
4. Transfer the aqueous phase (top) containing the partially purified RNA to a fresh 15 mL screw cap tube.
• Be careful to get as much of the aqueous layer without any contamination from the white interface layer or organic layer on the bottom.
• Immediately proceed to the final RNA purification steps below, which can be performed back at the bench.

### Final RNA Purification

1. Preheat 100 μL of Elution Solution per sample to 95°C in the heat block (this is conveniently set up when the samples are being lysed).
2. Add 1.9 mL Binding Buffer to the aqueous phase recovered previously and mix thoroughly by vortexing.
• It is convenient to add 950 μL twice using the P1000 pipettor.
3. Add 1.25 mL 100% ethanol to each sample and mix thoroughly by vortexing.
• It is convenient to add 625 μL twice using the P1000 pipettor.
4. Apply 700 μL of each sample mixture to a Filter Cartridge assembled in a Collection Tube.
• Spin for 1 minute at 10,000 rpm.
5. Repeat the previous step with 700 μL applications of the sample mixture until all of the lysate has been passed through the filter.
• Note that the RNA is now bound to the filter in the Filter Cartridge.
6. Wash the filter by pipetting 700 μL Wash Solution 1 on to the filter
• Spin for 1 minute at 10,000 rpm.
7. Wash filter with 500 μL Wash Solution 2/3.
• Spin for 1 minute at 10,000 rpm.
8. Wash filter with a second application of 500 μL Wash Solution 2/3.
• Spin for 1 minute at 10,000 rpm.
• Centrifuge again for 1 minute to remove excess wash solution from the filter.
9. Carefully Transfer the Filter Cartridge to a fresh 2 mL Collection Tube.
• Elute the RNA by applying 50 μL of Elution Solution (pre-heated to 95°C) to the center of the filter.
• Be careful that the solution gets onto the filter without actually poking it with the pipette tip.
• Note that the protocol calls for eluting with 25-50 μL of Elution Solution and we are using 50 μL.
10. Centrifuge for 1 minute. DO NOT DISCARD THE FLOW-THROUGH BECAUSE IT NOW CONTAINS YOUR RNA.
11. Repeat with a second application of 50 μL of Elution Solution (pre-heated to 95°C) to the center of the filter.
12. Centrifuge for 1 minute, keeping the flow-through. The filter cartridge can now be discarded because your RNA is now at the bottom of the tube.
13. Place the tubes on ice. The RNA can be stored at -80°C, or you can proceed immediately to the Turbo DNase Treatment step below.

## Turbo DNase Treatment

1. Assemble the Turbo DNase digestion reaction. To each tube add:
Reagent                   Volume
10X Turbo DNase Buffer    10   μL (mix and spin)
Turbo DNase (2 units/μL)   1.5 μL (3 units)

1. Flick and spin and incubate 30 minutes in the 37°C water bath.
2. Add an additional 1.5 μL (3 units) of Turbo DNase enzyme, flick and spin, and incubate for an additional 30 minutes in the 37°C water bath.
3. Treat with 0.2 volumes (22.6 μL)of DNase Inactivation Reagent.
• Resuspend the DNase Inactivation Reagent by vortexing. Pipet 22.6 μL of the Inactivation Reagent, being careful to get some of the beads into your pipet tip.
• Mix by vortexing and allow the reaction to stand at room temperature for 5 minutes.
• Centrifuge 3 minutes at 10,000 rpm at room temperature in the microcentrifuge.
• Transfer the supernatant to a fresh 1.5 mL tube, being careful to not get any of the beads in the pipet tip. If you do, pipet them back out into the sample. Spin again and try again.
• Store the purified RNA at -80°C.