DjJ Applications in Genetics
Experimental
Protocol
Each of the 3' -fluorescein-labeled anchor probes binds with a distance of one base
5' to their corresponding detection probes. All fluorophore-Iabeled probes were synthesized and purified by reverse-phase HPLC by TIB MOLBIOL, Berlin, Germany.
The analysis on the LightCyder was performed in a reaction volume of 20 fil with
40 ng of genomic DNA and a master mix of the following composition:
Volume [1l11
IFinal]
LightCycler-DNA Master Hybridization Probes
MgCl2 (25 mM)
Primers (3 j.1M each)
Probes (4 j.1M each)
H20 (PCR grade)
Total master mix volume per reaction
2.0
1.2
2.0
2.0
10.8
18.0
Ix
2.5mM
0.3 j.1M each
0.4 j.1M each
After loading the samples into the glass capillary cuvettes, the DNA template was
added (2 fil=40 ng) and the capillaries were sealed, briefly centrifuged, and then
placed into the LightCyder rotor. For amplification the following thermocyding
protocol was used:
• Denaturation at 95°C for 30 s
• Amplification
Parameter
Value
Cycles
Type
Target temperature rOC]
Incubation time Is]
Temperature transition rate [OC/s)
Acqwsition mode
Gains
• Melting Curve Analysis
45
Quantification
Segment I
Segment 2
95
60
0
6
20
20
None
Single
FI=I; F2=10; F3=30
Parameter
Value
Cycles
Type
Melting curve
Segment I
Segment 2
Target temperature [0C)
95
35
Incubation time [sl
30
150
Temperature transition rate [OC/s]
20
20
Acquisition mode
None
None
Gains
FI= I ; F2=10; F3=30
Segment 3
72
6
3
one
Segment 3
75
0
0.1
Cont.