5.6. Second PCR with Indexed Primers

This step adds a dual unique DNA/RNA UDP index combination to each 1st PCR Product generated in the previous step, as well as universal flanking P5 and P7 sequences needed for cluster formation on the Illumina NGS flow cell (Outline).

The Index Primer Plate in the kit contains a unique combination of Forward and Reverse DNA/RNA UDP index primers in each of 96 wells for both 24 and 96 sample kits. Each well contains 20 μL of unique index primer combination (10X concentration), which is enough to run x4 2nd PCR reactions (5 μl per 50 μl reaction). One unique index primer combination should be used for each sample (triplicate samples have different indexes). If you run a small number of samples (e.g., 2-3), consider running the 2nd PCR step in triplicate using different index primers to avoid low complexity index sequencing in the NGS step. Always record the well coordinates of index primers used for each sample to avoid problems in the NGS sequences deconvolution step. Prior to use, thaw the Index Primer Plate, briefly centrifuge the plate to collect droplets to the bottom of the wells, remove the seal slowly (to prevent cross-contamination), and aliquot 5 μl for each sample (45 μl) as described below. Seal the plate with a new sealing film and store at +4 °C (for immediate use) or -20°C (for prolonged storage). See Appendix. List of Dual DNA/RNA UDP Indexes for the sequences of each Forward and Reverse Index combination.

*If you have a kit with dried index plates, please follow the protocol here. All other library prep steps are the same.

Arrangement of the index pairs in the supplied Index Primer Plate.

1. Prepare enough of the 2nd PCR Master Mix, following the formulation below for all samples and controls, plus 5% extra volume of all components:
    
   

   2nd PCR Master Mix Component	Volume per sample, µl
   PCR Buffer, 5X	10
   dNTP Mix	1
   Water	33.5
   DNA Polymerase	0.5
   Total	45

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

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

3. Aliquot 45 µl of the 2nd PCR Master Mix into selected wells of the new 96-well plate and add 5 μl of unique index primer combination in each well from the Index Primer Plateprovided in the kit. To avoid index-to-index contamination, add index primers from the Index Primer Plate using a new tip for each well. Seal the Index Primer Plate with a new sealing film and store at +4 °C for up to 3 months for future use.

4. Spin down, then remove the seal from the 1st PCR Plate. Transfer 2 µl of the 1st PCR products to each of the Index Primer PCR reactions prepared in step 3. To avoid mistakes, ensure that samples in the 1st PCR plate are arranged in the same format as the Index PCR Primer pair mixes in the Index Primer PCR Reaction Plate (e.g. Sample 1A is aliquoted to well 1A). We recommend that you immediately record the Sample name and well number/index 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 Primer PCR Reaction	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.