TaqMan SNP Assay
Materials
- TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
- TaqMan® Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
- StepOnePlus Real-Time PCR System (Applied Biosystems).
- PC/Laptop with StepOnePlus Software installed.
- MicroAmp Fast 96-Well Plates.
- Optical Plate Covers.
- Nuclease-Free Water
- Sample Genomic DNA (extracted as described in Blood DNA Extraction)
- Postive control(s) (Minor allele) (optional, but recommended)
Protocol
- Dilute gDNA samples to ng/ul.
- Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice.
- Make up Reaction Master mix (see table below).
Table 1. Reaction Mix for full 96 well plate
| Component | 1X volume | 96X Volume (+ 10% excess) |
|---|---|---|
| TaqMan® Genotyping Master Mix (2X) | 5.00 ul | 528.0 ul |
| TaqMan® Genotyping Assay (40X) | 0.25 ul | 26.4 ul |
| 5.25 ul | 554.4 ul |
- Reaction Mix (5.25 ul) added to each well (see Table 2.)
- Diluted gDNA samples each added to a single well (see Table 2.)
Table 2. for full 96 well plate
| Component | 1X volume | 96X Volume (+ 10% excess) |
|---|---|---|
| Reaction Master Mix | 5.25 ul | 554.4 ul |
| gDNA samples | 4.75 ul | 501.6 ul |
| 10 ul | 1056 ul |