STANDARD ECLIPSE PROTOCOL
1 – DNA PLATES PREPARATION:
Dispense 10ng DNA per well to TaqMan PCR plates (DRIED DNA). Remember to leave some wells empty in order to have NTC (No Template Controls).
The reaction can either be done in 96 wells-plate or in 384 wells-plate format.
Positive controls of known genotype (if available) can be added on plates along with the samples.
2 – PROBES and PRIMERS:
Primers and Probes solutions must be kept frozen at -20°C.
2.1 - Probes :
Probes come in 20X Mix. Quenchers are TET and FAM.
NB: Keep all probes in the dark as much as possible.
2.2 - Primers :
Primers come in 20X Mix.
3 – MASTERMIX:
You have to prepare it prior to from the MGB Eclipse PCR Reagent Kit for SNPs provided by Sigma-Aldrich or from Nanogen.
Mix for preparation of 2X MasterMix for one 384 wells-plate:
MGB Eclipse Premix 940 µL
JumpStart Taq DNA Polymerase (2.5unit/µL) 60 µL
TOTAL VOLUME 1000 µL
4 – PCR MIX(384 wells-plates):
MasterMix (2X) 1000 µL
MGB Eclipse Probe Mix (20X) 100 µL
MGB Eclipse Primer Mix (20X) 100 µL
H2O D 800 µL
Dried DNA (10ng)
FINAL VOLUME 5 µL
5 – THERMOCYCLING:
50ºC for 2 minutes (if using Master Mix with UNG)
95ºC for 2 to 10 minutes (depending on the enzyme you use)
95ºC for 5 seconds
50 cycles 58ºC for 20 seconds
76°C for 30 seconds
10°C forever
6 – MELT CURVE:
The melt curve is done using the ABI 7900HT sequence detection system.
10% Ramp Rate
95ºC for 15 seconds
30ºC for 15 seconds
80ºC for 15 seconds
Data must be collected during ramp and at 80°C incubation.
Once the melt curve is done, the dissociation curve data are exported in text format. That file is then imported in Dynascore in order to analyze result.
NB: Remember to remove ROX as passive reference if necessary.
7 – RESULTS:
The melt curves data are analyzed using the DynaScore software provided by DynaMetrix.