TaqMan SNP Assay: Difference between revisions

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 == Materials ==
-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).
+*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'')
-TaqMan Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
-MicroAmp Fast 96-Well Plates.
+== 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.)
-Optical Plate Covers.
-StepOnePlus Real-Time PCR System (Applied Biosystems).
+'''Table 1. Full 96 well plate'''
+{| class="wikitable"
-PC/Laptop with StepOnePlus Software installed
+|-
+! Component
+! 1X volume
-== Protocol ==
+! 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
+|}