Amplicon PCR

Start with this section if using DNA only or both RNA and DNA inputs per sample. If using both RNA and DNA inputs, add purified DNA from each sample.

This step uses the custom primer pool(s) to amplify targets of interest from each sample to produce the amplicons that will be tagmented.

This portion of the protocol is designed to use two primer pools to tile across a genome or large target region. You can also use this protocol with one primer pool.

• IPM (Illumina PCR Mix)
• Primer Pool 1 (PP1) (User-designed and sourced)
• Primer Pool 2 (PP2) (User-designed and sourced)
• Nuclease-free water
• 15 ml tube
• 96-well PCR plates (2)
• Microseal 'B' adhesive seal
1. Prepare the following consumables:
• PP1—Keep on ice until use.
• PP2—Keep on ice until use.
• IPM—Invert to mix. Keep on ice until use.
2. Save the following IMAP PCR program on the thermal cycler:
• Choose the preheat lid option at 105°C
• Set the reaction volume to 25 µl
• 98°C for 3 minutes
• 35 cycles of:
• 98°C for 15 seconds
• 63°C for 5 minutesOpt Option
• Hold at 4°C
3. Label two new PCR plates PP1 and PP2.

The plates represent two separate PCR reactions on each sample in the CDNA1 plate. Alternatively, you can set up both sets of PCR reactions in a single PCR plate.

This step in the PCR cycling program can be optimized. For example, you can decrease the combination 63°COpt Option anneal/extension temperature to better match the annealing temperatures of the PCR primer pools to create a true annealing step in the cycle. You can also insert an additional extension step at 72°C into the cycle. You can optimize both temperature and time, depending on your targets and amplicons.

1. In two 1.7 ml tubes, combine the following reagents to prepare PCR 1 Master Mix and PCR 2 Master Mix. Multiply each volume by the number of samples.

Reagent overage is included to account for small pipetting errors.

Reagent

PCR 1 Master Mix (µl)

PCR 2 Master Mix (µl)

IPM

15

15

PP1

4.3

N/A

PP2

N/A

4.3

Nuclease-free water

4.7

4.7
2. Add 20 µl PCR 1 Master Mix to each well of the PP1 plate.
3. Proceed as follows:
• RNA only: Add 5 µl cDNA from each well of the CDNA1 plate to the corresponding well of the PP1 plate.
• DNA only: Add 5 µl purified DNA from each sample to a well of the PP1 plate.
• RNA and DNA: Add 2.5 µl cDNA from each well of the CDNA1 plate to the corresponding well of the PP1 plate, followed by 2.5 µl of purified DNA from the same sample to the corresponding well of the PP1 plate. Each well of the PP1 plate should contain 2.5 µl of cDNA and 2.5 µl of DNA from the same starting sample, plus the PCR 1 Master Mix.
4. Add 20 µl PCR 2 Master Mix to each well of the CDNA2 plate.
5. Proceed as follows:
• RNA only: Add 5 µl cDNA from each well of the CDNA1 plate to the corresponding well of the PP2 plate.
• DNA only: Add 5 µl purified DNA from each sample to a well of the PP2 plate.
• RNA and DNA: Add 2.5 µl cDNA from each well of the CDNA1 plate to the corresponding well of the PP2 plate, followed by 2.5 µl of purified DNA from the same sample to the corresponding well of the PP2 plate. Each well of the PP2 plate should contain 2.5 µl of cDNA and 2.5 µl of DNA from the same starting sample, plus the PCR 2 Master Mix.
6. Seal and shake at 1600 rpm for 1 minute.
7. Centrifuge at 1000 x g for 1 minute.
8. Place in the preprogrammed thermal cycler and run the IMAP PCR program.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at ‑25°C to ‑15°C for up to 3 days.