DIjI Application.s in Genetics
LightCycier PCR
The following mix was used for melting point analysis with artificial complements:
LightCycier FastStart DNA Master Hybridization Probes
MgC12 (25 mM)
Probes (10 j.lM)
H20 (PCR grade)
Complement (10 fLM)
Total volume
Volume [!J.l[
Final
2
1.6
0.4+0.4
14.6
I
20
Ix
3.0mM
0.2j.lM
0.5 fLM
The following master mix was used to determine the methionine/valine polymorphism at position 129 of the human prion protein with hybridization probes:
LightCycler Fa. stStart DNA Master Hybridization Probes
MgClz (25 mM)
Forward primer (10 fLM)
Reverse primer (0.25 fLM, 0.5 fLM, 1.25 j.lM, 2.5 j.lM)
Probes (10 fLM)
H20 (PCR grade)
Total volume
Volume [!J.l)
Final
0.8
0.5
2
0.2+0.2
2.8
7.5
Ix
3mM
0.5 fLM
0.05-0.5 j.lM
0.2fLM
A total of 7.5 fJl of master mix and 2.5 fil of DNA (concentration not determined)
were added to each glass capillary placed in precooled adaptors. Sealed capillaries
were centrifuged with the adaptors (lOOOx g for 1 min) and placed into the LightCycler rotor.
The following PCR protocol was used for amplification:
• Denaturation of DNA and activation of FastStart polymerase for 10 min at 95°C
• Amplification
Parameter
Value
Cycles
45
Type
Quantification
Segment 1
Segment 2
Segment 3
Target temperature [0C)
95
5S
72
Incubation time [s]
0
5
10
Temperature transition rate [OC/s]
20
20
20
Acquisition mode
None
Single
None