Difference between revisions of "TaqMan SNP Assay"

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# Dilute gDNA samples to 20ng/ul approx.
 
# Dilute gDNA samples to 20ng/ul approx.
 
# 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 (TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X)).
+
# 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.)
 
# Add Reaction Mix (5.25 ul) added to each well (Table 1.)
 
# Diluted gDNA samples each added to a single well (Table 1.)
 
# Diluted gDNA samples each added to a single well (Table 1.)
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|-
 
|-
 
|gDNA Sample (20ng/ul)
 
|gDNA Sample (20ng/ul)
|4.50
+
|4.50 ul
|501.6
+
|501.6 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

  1. Dilute gDNA samples to 20ng/ul approx.
  2. Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice.
  3. Make up Reaction Master mix (TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X): see table below).
  4. Add Reaction Mix (5.25 ul) added to each well (Table 1.)
  5. 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