Difference between revisions of "TaqMan SNP Assay"
From Bridges Lab Protocols
| Line 14: | Line 14: | ||
== Protocol == | == Protocol == | ||
| − | # Dilute gDNA samples to | + | # Dilute gDNA samples to ng/ul. |
# 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 (see table below). | # Make up Reaction Master mix (see table below). | ||
| − | + | '''Table 1. Reaction Mix for full 96 well plate''' | |
{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
| Line 37: | Line 37: | ||
!5.25 ul | !5.25 ul | ||
!554.4 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''' | ||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | ! 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 | ||
Revision as of 16:28, 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 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
Table 2. for full 96 well plate
|
