Difference between revisions of "TaqMan SNP Assay"
From Bridges Lab Protocols
| (21 intermediate revisions by the same user not shown) | |||
| Line 3: | Line 3: | ||
*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems). | *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). | *StepOnePlus Real-Time PCR System (Applied Biosystems). | ||
*PC/Laptop with StepOnePlus Software installed. | *PC/Laptop with StepOnePlus Software installed. | ||
| Line 9: | Line 9: | ||
*Optical Plate Covers. | *Optical Plate Covers. | ||
*Nuclease-Free Water | *Nuclease-Free Water | ||
| − | *Sample Genomic DNA | + | *Sample Genomic DNA (extracted as described in [[Blood DNA Extraction]]) |
*Postive control(s) (Minor allele) (''optional, but recommended'') | *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 |
