The Ambion 5X T7 MEGASCRIPT kits are on the third shelf of Freezer #1. Each kit has enough enzyme and dNTP for two 96-well plates’ worth of reactions.

Synthesizing dsRNA

1. Wipe down the work bench with 70% ethanol.

2. Produce the following master mix for each 96-well plate you wish to do an IVT reaction on. We use the Ambion 5X T7 MEGASCRIPT Kit; AMB 1334-5.

• IVT reaction buffer is extremely high in salt, so make sure the salt is fully dissolved before using the buffer. ½ IVT reactions produce around 30-60µg of dsRNA, enough to produce about 60 384-well assay plates.

3. Dispense 12µL of master mix into each well of a 96-well PCR plate. Use a multichannel pipette and reservoir to speed this process along.

4. Transfer 8µL of amplified PCR product to its corresponding well.

5. Seal the plate using the PlateLoc. Mix by inverting. Spin down for 1 minute at 2,000rpm. Label and date the plate.

6. Incubate the IVT reaction overnight (~16 hours) at 37°C

7. Make a 1:5 dilution of the Turbo DNAse from the Ambion kit using nuclease-free water. Make about 500µL if you have a full 96-well plate(100µL DNAse, 400µL water). Add 5µL to each well using a multichannel pipette.

8. Incubate at 37°C for 45 minutes. During this time, wipe down workbench with 70% ethanol.

9. Set up the vacuum manifold for IVT clean up.

10. Add 175µL of nuclease-free water to each well of a vacuum filter plate (currently using Millipore Filter plate MSNU 03050), then transfer the samples to the vacuum filter plate, mix by pipetting, and place the lid on top of the plate.

11. Vacuum filter on a vacuum manifold for 45 minutes at 15-20 PSI. The vacuum manifold may be finicky and require pressing down on the plate lid to get a good vacuum seal. Check on the pressure occasionally throughout the 45 minutes, as it may fall out of range and need to be readjusted.

12. After vacuuming, ensure that the wells are completely dry. Add 75µL of nuclease-free water to each well.

13. Place the lid of the filter plate back on and shake for 45 minutes using the orbital shaker set to the third circle. The orbital shaker is in Bay 1, across from the gel electrophoresis rigs.

14. Transfer the dsRNA from the filter plate into either a fresh 96-well PCR plate or the 96-well plates the IVT reactions were prepared in. Label and date the plate.

15. Use the Nanodrop to measure the concentrations of the dsRNA to see if IVT worked. Use the “Other” sample setting by changing it in the drop-down menu from “DNA-50” and adjusting the concentration factor to 45 ng/uL.

• Look at the curve and the concentration. Most dsRNA should fall in a concentration range of 1000-3000 ng/uL. The 260/280 ratio should be about 1.9-2.2. Re-measure any dsRNA with a concentration higher than 3000 ng/uL to double check.

16. Run 0.5µL of dsRNA on a 0.7% agarose gel and determine quality.

• Mix 0.5uL dsRNA with 4.5uL nuclease-free water for the 5uL needed to run a gel. Add 1uL of loading buffer, and take 5uL of the resulting mix to load the gel.

Store dsRNA plates in -80 freezers. Make sure all plates are labeled and dated.

Note: For “custom IVT” distributions, we send both the dsRNA (often normalized and sometimes aliquoted to assay-ready plates, depending on the user request) and the PCR templates that we cherry-picked and used to make the dsRNA. Including the PCR templates in the shipment in addition to the dsRNA itself allows the users to cherry-pick one or a few templates and make more dsRNA for those designs at low-throughput, for example for their follow-up studies.

Move onto the Normalization and Re-array protocol.