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TaqMan SNP Assay

984 bytes added, 16:39, 16 November 2015
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== Materials ==
*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X)(Applied Biosystems).TaqMan *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 platesPlates.*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'''
{| 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
|}
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