( embrane which physically captures the R...)Total RNA Purification from Cultured Cells Using the ABI PRISM 6700 Automated Nucleic Acid Workstation and Total RNA Lysis Reagents

embrane which physically captures the RNA, a very low dead volume drip director, and an aerosol guard to prevent well-to-well cross-contamination. The RNA captured on the membrane is washed to remove any traces of cellular components and then re-solubilized and eluted from the membrane in a PCR compatible buffer.

Protocols

The purified total RNA can be used as follows: (Figure 1)

One or two daughter plates can be created at user-defined dilutions.

cDNA can be prepared from the purified RNA, using the 6700 workstations Peltier temperature control feature.

Up to four output plates can then be prepared, containing standards, controls, reagents for 5' nuclease assay or other nucleic acid-based assay reagents. A sample of the originally purified nucleic acid archive, dilution archive or cDNA archive, is added to the output plate.

The output plates are optionally heat-sealed with an optically clear film and are ready for analysis using the ABI PRISM 7700 or GeneAmp 5700 Sequence Detection System.

Archive tray covers are optionally placed on the purified nucleic acid archive, dilution archive or cDNA archive.

Definition of Terms

2X lysis reagent: Lysis Reagent, Nucleic Acid Purification (P/N 4305895)

lysis buffer: 1:1 mixture of P/N 4305895 and phosphate buffered saline, Ca²⁺/Mg²⁺ free

Methods

Lysis of Suspension or Adherent Cells Recommended input ranges for Applied Biosystems total RNA chemistry: 104-106 cells/well. Recommended maximum lysate volume for the 6700 workstation: 250 μL lysed cells/well in a 300 μL cell culture plate.*

(*See Ordering Information for recommended cell culture plates.)

Lysis and Purification of RNA from Suspension Cell C
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