TaqMan SNP Assay: Difference between revisions

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== Protocol ==
== Protocol ==
# Dilute gDNA samples to 1-5 ng/ul.
# 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).




Master Mix for full 96 well plate
'''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

  1. Dilute gDNA samples to ng/ul.
  2. Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice.
  3. 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
  1. Reaction Mix (5.25 ul) added to each well (see Table 2.)
  2. Diluted gDNA samples each added to a single well (see Table 2.)

Table 2. for full 96 well plate

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
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