TaqMan SNP Assay: Difference between revisions
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== Materials == | == Materials == | ||
TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X). | *TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems). | ||
*TaqMan® Genotyping Master Mix (supplied at 2X)(Applied Biosystems). | |||
MicroAmp Fast 96-Well Plates. | *StepOnePlus Real-Time PCR System (Applied Biosystems). | ||
Optical Plate Covers. | *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 == | == 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''' | |||
{| class="wikitable" | |||
|- | |||
! 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 | |||
|} | |||
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 |