Bio-rad Aurum™ Total RNA 96 Kit Manual do Utilizador descarregar pdf (Página 17)

B2. 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.

 B3. Add250µlof70%isopropanol(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.

Yeast

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

onpage13.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. Place an RNA binding plate on top of a waste tray or a pipet tip box lid.

2. Transfer the lysate from each well of the 96-well plate into a

corresponding well of the RNA binding plate.

3. Centrifuge for 2 min. Discard the filtrate from the waste tray and replace

the RNA binding plate on top of the same waste tray.

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 do Utilizador Página 17