In this amplification step, flanking sequences for the P5 and P7 Illumina adapters are added to the 3’ L1 DNA product using the Adapter P5 Primer and one (from the 24) P7 Indexed Primers. Samples with different P7 Indexed Primers can be deconvoluted based on the unique index sequence when loaded in the same lanes of the Illumina Sequencing Cell (see Appendix B: Structure of the Adaptor Sequences of the 3’-Amplicon). As a result, the DNA products with different indexes can be mixed together for cluster generation on the Illumina NGS Sequencing Cell.
- Use 2 µl of the Second Nested PCR product from Step 5 for the final P5/P7 PCR reaction below.
- For each indexing reaction, use the appropriate Indexing Primer (one of the P7 Ind1-24 primers). Samples that will be sequenced in the same lane of the Illumina cell must have different P7 Index Adaptors. Be certain to keep track of which P7 Index Adaptor was used to amplify each sample so the sequencing results can be deconvoluted to assess the specific reads for each sample.
- Prepare the reaction mix on ice in a 50 μl thin wall PCR tube:
Component Volume DNA sample from 2nd PCR (Step 5) 2 µl HF DNA Polymerase Buffer (5X) 10 µl dNTP Mix (50X) 1 µl P7 Indexed Primer 1-24 (as appropriate) 1 µl P5 Primer 1 µl PCR-Grade Water 34.5 µl HF DNA Polymerase (100X) 0.5 µl Total 50 µl
- Mix gently and centrifuge briefly to collect droplets.
- Amplify Indexed-3’L1H-DNA products using the following PCR program:
98°C, 60 seconds 1 cycle 98°C, 15 seconds
62°C, 15 seconds
72°C, 30 seconds
10 cycles 72°C, 60 seconds 1 cycle 4°C, ∞ 1 cycle
- Analyze the P7-Index-3’L1H-DNA-P5 product on a 3% Agarose TAE gel. Cast a 3% agarose gel with 1X TAE buffer and 400 ng/ml EtBr (60 µg). Mix 5 µl of each PCR product from the Step 6 amplification with 5 µl of 2X loading buffer (e.g. 50% sucrose with Bromophenol Blue tracking dye) and load onto the gel. In at least 1 well, include a 100-bp DNA ladder.
- Run gel at 5 V/cm until the 100-bp band is about an inch from the bottom. PCR products should appear as a smear of approximately 250-500 bp with the brightest region around 350-450 bp (see Example Gel Data image).
Optional Analysis Step: To validate the quality of the Indexed-3’L1H-DNA, use the “Library Validation for Illumina NGS by TA Cloning” procedure described in Appendix A. This may be useful to check if you are having trouble getting good sequencing runs.
Need more help with this?