6.5. Second PCR with Indexed Primers

This step adds a dual unique DNA/RNA UDP index combination to each Anchored PCR Product generated in the previous PCR with Anchor Primers step as well as universal flanking P5 and P7 sequences needed for cluster formation on the Illumina NGS flow cell (Outline).

The Index PCR Plate in the kit contains a unique combination of Forward and Reverse DNA/RNA UDP index primers in each well. The primers have been dried onto the bottom of each well and will be dissolved when the PCR reaction mix with the sample is added. One well should be used for each sample (triplicate samples are different samples) being sequenced. See Appendix D. List of Dual DNA/RNA UDP Indexes for the sequences of each Forward and Reverse Index combination.

For the 24 samples, use any two 2 lines (e.g., A1-B12) or 3 columns (e.g., 1A-3H) of well for the samples. If you are processing less than 24 samples, you can use scissors to cut the desired number of wells from the index plate (e.g., cut 1-2 columns for 8 samples), then store the rest of the plate for later use.

*NOTE: If you run samples on a cut-out portion of the plate, be sure to reference the well location relative to the whole plate when entering the sample names at each plate index.

Arrangement of the index pairs for 96 samples

1. Prepare enough of the Index PCR Master Mix, following the formulation below for all samples and controls plus 5% extra volume of all components:
    
   

   Index PCR Master Mix Component	Volume per sample, µl
   PCR Buffer, 5X	10
   dNTP Mix	1
   Water	38.5
   DNA Polymerase	0.5
   Total	50

*Note: If you are using a multichannel pipette, aliquot the master mix in a tube strip to minimize pipetting steps.

2. Gently vortex the Index PCR Master Mix, and spin down briefly to collect droplets. Remove the plate seal from the Index PCR Plate. Set up the Index Primer PCR Reactions as follows:

3. Aliquot 50 µl of the Index PCR Master Mix into appropriate wells of the 96-well Index PCR Plate (or cut-off portion of the plate) provided in the kit. To avoid index- to-index contamination, add Index PCR Master Mix using a new tip for each well.

4. Spin down, then remove the seal from the Anchor Primer PCR plate (plate from PCR with Anchor Primers step). Transfer 2 µl of Anchored PCR products to each of the Index Primer PCR reactions on the Index PCR Plate. To avoid mistakes, ensure that samples in the Anchor Primer PCR plate are arranged in the same format as the Index PCR Primer pair mixes in the Index PCR Plate (e.g. Sample 1A is aliquoted to well 1A). We recommend that you record the Sample name and well number (e.g., Sample 1 in well 1A) for all the samples, including the positive control. This will help minimize mistakes in the NGS deconvolution step.
    
   

   Component	Volume per sample, µl
   Anchored PCR Product (step above)	2
   Index PCR Master Mix (above)	50
   Total	52

5. Seal the plate with new adhesive film and spin down to collect droplets.

6. Load the plate in the thermal cycler, and run the following program:
    
   

   Temperature	Time	Cycles
   98°C	30 sec	1
   98°C	20 sec	8
   65°C	10 sec
   72°C	30 sec
   72°C	30 sec	1
   4°C	∞	1

!Stopping Point in procedure: PCR products may be stored at 4°C overnight.