TAQMAN ASSAY A.0779
1 - DNA PLATES PREPARATION :
Dispense 10 ng DNA per well, in Taqman PCR plate (DRIED DNA)
The reaction can either be done in 96 wells-plate or in 384 wells-plate format.
Positive controls of known genotype (if available) as well as NTCs (no template controls) are
added on plates along with the samples.
2 - PROBES and PRIMERS PREPARATION :
Primers and Probes mother solutions must be kept frozen at -20°C
2.1 - Probes :
Probes must be at 100µM.
NB : Keep all probes in the dark.
Quenchers are TET and FAM.
2.2 - Primers :
Primers must be at 100µM
3 - PCR1 MIX :
3.1 - 96 wells-plate :
Buffer (10X)
dNTPS (2mM each)
MgCl2 (50mM)
Forward Primer (100M)
Reverse Primer (100M)
H2O 
Taq Platinum (5U/µL)
Dried DNA (10ng)
FINAL VOLUME
3.2 - 384 wells-plate :
80
80
32
3.2
3.2
593.6
8
µL
µL
µL
µL
µL
µL
µL
Buffer (10X)
dNTPS (2mM each)
MgCl2 (50mM)
Forward Primer (100M)
Reverse Primer (100M)
H2O 
Taq Platinum (5U/µL)
Dried DNA (10ng)
8
µL
FINAL VOLUME
200
200
80
8
8
1484
20
µL
µL
µL
µL
µL
µL
µL
5
µL
4 - THERMOCYCLING :
50ºC for 2 minutes (if using Master Mix with UNG)
95ºC for 10 minutes
95ºC for 30 seconds
20 cycles
58°C for 30 seconds
72ºC for 30 seconds
72ºC for 5 minutes
16°C forever
5 - PCR2 MIX :
3.1 - 96 wells-plate :
3.2 - 384 wells-plate :
Master Mix (2X)
Forward Primer (100M)
Reverse Primer (100M)
Probes VIC (100M)
Probes FAM (100M)
H2O 
400
8
8
1.6
1.6
180.8
µL
µL
µL
µL
µL
µL
Master Mix (2X)
Forward Primer (100M)
Reverse Primer (100M)
Probes VIC (100M)
Probes FAM (100M)
H2O 
1000
20
20
4
4
152
µL
µL
µL
µL
µL
µL
MIX
Half-diluted PCR product
6
2
µL
µL
MIX
Half-diluted PCR product
3
2
µL
µL
FINAL VOLUME
8
µL
FINAL VOLUME
5
µL
6 - THERMOCYCLING :
50ºC for 2 minutes (if using Master Mix with UNG)
95ºC for 10 minutes
40 cycles
95ºC for 15 seconds
62ºC for 1 minute
10°C forever
7 - RESULTS :
The plate is then read on the ABI 7900HT sequence detection system.
The SDS software graphs the results of allelic discrimination run on a scatter plot of Allele 1 Rn
versus Allele 2 Rn.
The software represents each well of the plate as a spot on the graph.
The genotypic segregation is displayed in the allelic plot.
The plot contains four distinct clusters, which represent the NTCs (no template controls) and
three possible genotypes that cluster along the horizontal, vertical and diagonal axes and
represent the Allele 1, Allele 2 and Allele 1/Allele 2 respectively.
This variation is due to differences in the extent of PCR amplification.
The data are then exported in text format for further analysis.