Bio-rad Aurum™ Total RNA 96 Kit Instrukcja Użytkownika Pobierz pdf (Strona 13)

Section 7

Vacuum Protocol

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

Section 6, “Vacuum Manifold Setup and Use With 96-Well Plates” 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 Sample Types” share a common protocol.

Cultured Cell Lines

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

on page 10.

 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 10. 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.

 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.

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Bio-rad Aurum™ Total RNA 96 Kit Instrukcja Użytkownika Strona 13