TaqMan SNP Assay: Difference between revisions
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*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. | ||
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*Optical Plate Covers. | *Optical Plate Covers. | ||
*Nuclease-Free Water | *Nuclease-Free Water | ||
*Sample Genomic DNA (extracted as described ) | *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 | |||
|} | |||