Mullins Molecular Retrovirology Lab

  • Department of Microbiology
  • School of Medicine
  • University of Washington
University of Washington/Fred Hutch Center for AIDS Research

Agilent Bioanalyzer RNA Samples

Preparing Gel-Dye-Mix:

  1. Put 400 µl RNA gel matrix (red dot ●) and 4 µl RNA dye concentrate (blue dot ●) in a 1.5ml tube.

  2. Vortex well. Transfer to spin filter.

  3. Spin 10 minutes at 3000rpm. Protect from light. Store at 4°C. Use within 3 weeks.

Preparing ladder and samples:

  1. Denature 1.5 µl RNA ladder for 3 minutes at 70°C. Chill on ice. Spin.

  2. Mark 0.6 ml tubes “ladder” and “1” to “12”

  3. Pipette 5 µl sample buffer (white dot ○) in each tube.

  4. Pipette 1 µl denatured RNA ladder in tube marked “ladder”.

  5. Pipette 1 µl of each sample into the appropriately marked tubes.

  6. Vortex each tube for 5 seconds.

  7. Spin tubes for 10 seconds.

  8. Store on ice until ready to load samples.

Loading Gel-Dye Mix:

  1. Put chip on preparation stand.

  2. Pipette 9 µl gel-dye mix in circled G well

  3. Pull back syringe-plunger to 1ml mark.

  4. Put syringe tip around well to form tight seal.

  5. Press plunger to 0.2ml and hold for 30 seconds.

  6. Pull back plunger to the 1ml mark while maintaining the seal.

  7. Remove syringe from well.

  8. Pipette 9 µl gel-dye mix in other 2 G wells.

Loading the ladder and samples:

  1. Pipette the 6 µl of ladder-tube in ladder-well

  2. Pipette the 6 µl of each sample-tube in the 12 µl sample wells.

  3. Treat bioanalyzer-electrodes for 30 seconds (no longer) with ZAP.

  4. Treat bioanalyzer-electrodes for 30 seconds (no longer) with ddH2O.

  5. Dry bioanalyzer-electrodes for 10 seconds (no longer).

  6. Insert the loaded chip into the bioanalyzer within 5 minutes after loading.

Reagents:

Agilent 2100 Bioanalyzer Reagent and Chip Kits
5064-8284 DNA 500 LabChip kit. Kit contains 25 LabChips, syringe, spin filters, and reagents for the quantitative analysis of DNA from 25-500bp. $299
5064-8230 DNA 7500 LabChip kit. Kit contains 25 LabChips, syringe, spin filters, and reagents for the quantitative analysis of DNA from 100-7500bp. $299
5064-8231 DNA 12000 LabChip kit. Kit contains 25 LabChips, syringe, spin filters, and reagents for analysis of DNA from 100-12000bp. $299
5064-8229 RNA 6000 LabChip kit. Kit contains 25 LabChips, syringe, spin filters, and reagents for analysis of RNA samples. $399

For quantitation of RNA samples, an RNA 6000 ladder must be purchased from Ambion, #7152.