Difference between revisions of "TaqMan SNP Assay"
From Bridges Lab Protocols
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# Dilute gDNA samples to 20ng/ul approx. | # Dilute gDNA samples to 20ng/ul approx. | ||
# Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice. | # Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice. | ||
| − | # Make up Reaction Master mix (TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X)). | + | # Make up Reaction Master mix (TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X): see table below). |
# Add Reaction Mix (5.25 ul) added to each well (Table 1.) | # Add Reaction Mix (5.25 ul) added to each well (Table 1.) | ||
# Diluted gDNA samples each added to a single well (Table 1.) | # Diluted gDNA samples each added to a single well (Table 1.) | ||
| Line 37: | Line 37: | ||
|- | |- | ||
|gDNA Sample (20ng/ul) | |gDNA Sample (20ng/ul) | ||
| − | |4.50 | + | |4.50 ul |
| − | |501.6 | + | |501.6 ul |
|- | |- | ||
! | ! | ||
Latest revision as of 16:39, 16 November 2015
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 20ng/ul approx.
- Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice.
- Make up Reaction Master mix (TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X): see table below).
- Add Reaction Mix (5.25 ul) added to each well (Table 1.)
- Diluted gDNA samples each added to a single well (Table 1.)
Table 1. 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 |
| gDNA Sample (20ng/ul) | 4.50 ul | 501.6 ul |
| 10 ul | 1056 ul |
