Amplify DNA

This step adds the bisulfite-converted DNA samples to the plates. The samples are denatured and neutralized to prepare them for amplification.

• MA1
• RPM
• MSM
• 0.1 N NaOH
• 96-well 0.8 ml microplates (MIDI)
• BCD plate with Bisulfite-converted DNA samples
• Cap mats
1. Preheat the Illumina Hybridization Oven in the post-amp area to 37°C and allow the temperature to equilibrate.
2. Prepare the following consumables:

Item

Storage

Instructions

BCD

-25°C to -15°C

Thaw at room temperature. Vortex to mix.

MA1

Room temperature

Thaw at room temperature. Invert 10 times to mix, and then pulse centrifuge to eliminate bubbles and collect reagent at the bottom of the tube.

RPM

-25°C to -15°C

Thaw at room temperature. Invert 10 times to mix, and then pulse centrifuge to eliminate bubbles and collect reagent at the bottom of the tube.

MSM

-25°C to -15°C

Thaw at room temperature. Invert 10 times to mix, and then pulse centrifuge to eliminate bubbles and collect reagent at the bottom of the tube.
3. Apply an MSA4 barcode label to a new MIDI plate.
1. Add 20 µl MA1 to each well of the MSA4 plate.
2. Transfer 4 µl DNA sample from the BCD plate to the corresponding wells of the MSA4 plate.
3. Add 4 µl 0.1 N NaOH to each sample well of the MSA4 plate.
4. Seal the MSA4 plate with the 96-well cap mat.
• Orient the mat so that A1 on the cap matches A1 on the plate.
• Make sure that all 96 caps are securely seated in the wells.
5. Vortex at 1600 rpm for 1 minute, and then pulse centrifuge at 280 × g.
6. Incubate at room temperature for 10 minutes.
7. Remove the cap mat and set aside upside down in a safe location.
8. Add 68 μl RPM to each sample well of the MSA4 plate.
9. Add 75 μl MSM to each sample well of the MSA4 plate.
10. Reseal with the cap mat using the original orientation.
11. Vortex at 1600 rpm for 1 minute, and then pulse centrifuge at 280 × g.