STANDARD TAQMAN PROTOCOL T0
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 :
If probes come dried, resuspend at 100 µM, then dilute part of it at 5 µM (working solution).
NB : Keep all probes in the dark.
PROLIGO provides TAMRA probes quenchers using HEX and FAM whereas APPLIED
BIOSYSTEMS provides MGB or TAMRA probes using VIC and FAM.
2.2 - Primers :
Dilute primers at 10 µM (working solution)
3 -PCR MIX :
3.1 - 96 wells-plate :
3.2 - 384 wells-plate :
Universal Master Mix ABI (2X)
Forward Primer (10M)
Reverse Primer (10M)
Probes A (5M)
Probes B (5M)
H2O
Dried DNA (10ng)
4 µL
0.8 µL
0.8 µL
0.32µL
0.32µL
1.76µL
Universal Master Mix ABI (2X)
Forward Primer (10M)
Reverse Primer (10M)
Probes A (5M)
Probes B (5M)
H2O
Dried DNA (10ng)
2.5
0.5
0.5
0.2
0.2
1.1
µL
µL
µL
µL
µL
µL
FINAL VOLUME
8
FINAL VOLUME
5
µL
µL
4 - THERMOCYCLING :
50ºC for 2 minutes (if using Master Mix with UNG)
95ºC for 10 minutes
40 cycles
95ºC for 15 seconds
60ºC for 1 minute
10°C forever
NB : Some probes might not work with 40 cycles. Pay attention to comments to get the
appropriate number of cycles. However, you can always add cycles later.
5- 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.