Bio-rad Aurum™ Total RNA 96 Kit Manual de usuario descargar pdf (Pagina 14)

Yeast

Follow steps C1–C5, then continue with step 1 of “All Starting Cell Types”

onpage10.Ifstartingwithagrowblockofyeastculture(maximum2OD/

ml/well),centrifugethegrowblockat1,500xgfor10min.Decantthe

supernatant, and blot the block with paper towels.

C1. Prepare 100 ml lyticase dilution buffer:

1 M sorbitol

0.1 M EDTA, pH 7.4

 0.1%(v/v)b-mercaptoethanol

Equilibrate the buffer at 30°C before use.

C2. Resuspend up to 2 x 10

yeastcellsperwell(2OD/ml/well),adding

1mlof50units/mllyticaseinlyticasedilutionbufferequilibratedto30°C

to each well. Incubate for 10 min.

C3. Centrifuge at 1,500 x g for 5 min. Decant the supernatant.

 C4. Add350µloflysissolution(alreadysupplementedwith1%

b-mercaptoethanol) to each sample and pipet up and down several times

to mix thoroughly.

 C5. Add350µlof70%ethanol(notsupplied)toeachsampleandpipetup

and down to mix thoroughly. Make sure that no bilayer is visible and that

the viscosity is substantially reduced.

All Starting Cell Types

1. Set up the Aurum™ Vacuum Manifold and one RNA binding plate

according to “Manifold Wash Setup” instructions on page 7.

2. With the vacuum turned off and the regulator open, transfer the lysate

from each sample to a well of the RNA binding plate.

3. Turn the vacuum on and gradually increase the negative pressure

between –17 to –23 inHg over a 5–10 sec period by slowly closing

thevacuumregulator.Onceallwellshaveemptied,openthevacuum

regulator fully. Check that the regulator gauge reads approximately 0 inHg.

Note: Gradual application of negative pressure is required to ensure

uniform flow of lysates through all 96 wells of the RNA binding plate.

4. The low stringency wash solution is provided as a 5x concentrate. Add

4 volumes (240 ml) 95–100% ethanol to the low stringency

wash solution concentrate before initial use.

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Bio-rad Aurum™ Total RNA 96 Kit Manual de usuario Pagina 14