Bio-rad Aurum™ Total RNA 96 Kit Uživatelský manuál stáhnout pdf (Strana 18)

 5. Add700µloflowstringencywashsolutiontoeachwelloftheRNA

binding plate. Centrifuge for 2 min at 1,500 x g. Discard the low

stringency wash solution from the waste tray and replace the RNA binding

plate on top of the same waste tray.

6. The RNase-free DNase I is provided as a lyophilized powder. Reconstitute

theDNaseIbyadding250µl10mMTris,pH7.5(notsupplied,seenote

below) to the vial. Pipet up and down briefly to mix.

 7. Foreachwell(ofa96-wellplate)processed,mix2.5µlofreconstituted

DNaseIwith77.5µlofDNasedilutionsolution.Forone96-wellplate,

mix the entire contents of one vial of reconstituted DNase I with 7.75 ml

DNase dilution solution in a 15 ml sterile conical tube. Scale proportionally

ifprocessingmoreorlessthanonefullplateatatime.Add80µldiluted

DNase I to the membrane at the bottom of each well of the RNA binding

plate. Cover the plate with sealing tape and allow the digestion to

incubate at room temperature for 10 min.

 8. Add700µlofhighstringencywashsolutiontoeachwelloftheRNA

binding plate. Centrifuge for 2 min at 1,500 x g. Discard the high

stringency wash solution from the same waste tray and replace the RNA

binding plate on top of the tray.

 9. Add700µloflowstringencywashsolutiontoeachwelloftheRNA

binding plate. Centrifuge for 2 min at 1,500 x g. Discard the low

stringency wash solution from the waste tray and replace the RNA binding

plate on top of the same tray. Centrifuge for an additional 2 min at

1,500 x g.

10. Place the RNA binding plate on top of the provided collection plate.

 11. Pipette80µl(or40µl)

†

of the elution solution onto the membrane stack

at the base of each well of the RNA binding plate and allow 1 min for the

solution to saturate the membranes. Centrifuge for 2 min at 1,500 x g.

†

Note: Pipet40µlwhenisolatingtotalRNAfromsmallamountsof

starting material (<10 mg of tissue or 500,000 cells).

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.

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Bio-rad Aurum™ Total RNA 96 Kit Uživatelský manuál Strana 18