Bio-rad Aurum™ Total RNA 96 Kit Manual do Utilizador descarregar pdf (Página 16)

Note: Gradual application of negative pressure is required to prevent

sample spraying and cross-contamination.

The eluted total RNA samples in the sample collection plate can be

used immediately in downstream applications. Alternatively, the sample

collection plate can be sealed with sealing tape and stored at –20°C or

–80°C for later use.

Section 8

Spin Protocol

 PleasereadSection5,“BeforeUsingtheAurum™TotalRNA96Kit”before

proceeding.

Except for the first few steps that are specific for the starting sample types

(A,forculturedcelllines;B,forbacteria;andC,foryeast),theremaining

procedures within “All Starting Cell Types” share a common protocol.

Cultured Cell Lines

Follow steps A1–A3, then continue with step 1 of “All Starting Cell Types” on

page 13.

 A1. Fornonadherentcellcultures,rinsethecellswithPBSandtransferup

to 1 x 10

cells into each well of a 96-well microplate (not provided).

Centrifuge the plate at 300 x g for 5 min then aspirate the supernatant

from each well.

For adherent cell cultures, rinse the wells (each containing up to

1 x 10

cells)ofthegrowthvesseloncewithPBSandaspirate.

 A2. Add150µloflysissolution(alreadysupplementedwith

1%b-mercaptoethanol) to each well and pipet up and down several times

to lyse cells thoroughly.

 A3. Add150µlof70%ethanol(notsupplied)toeachwellandpipetupand

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

viscosity is substantially reduced.

Bacteria

FollowstepsB1–B3,thencontinuewithstep1of“AllStartingCellTypes”

on page 13. If starting with a grow block of bacterial culture (maximum 1

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.

 B1. Resuspendupto8x10

bacterialcellsperwell(1OD/ml/well),adding

100µlof500µg/mllysozymeinTE(10mMTris,1mMEDTA,pH7.5).

Incubate at room temperature for 5 min.

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