•
Methods Useful in Genetics and Oncology
lightCycier-PCR SYBR Green Master Mix for each 20 III reaction:
Volume f1I
Final
LC-DNA Master SYBR Green I
2
Ix
MgClz (25 mM)
1.6
3mM
Primers (10 IlM each)
1+ 1
0.5 11M each
H2O
12.4
Total master mix volume per reaction
18
18111 of master mix and 2 III DNA (25 ng/Ill) were added to each capillary. Sealed
capillaries were centrifuged and placed into the LightCyder rotor.
The following LightCyder-protocol was used for amplification of fragments
~90bp. For amplification of the fragments of 69bp and 76bp incubation time was
reduced to 5 s.
• Denaturation for 120 s at 95°C.
• Amplification
Parameter
Value
Cycles
Type
Target temperature [0C]
Incubation Time (s)
Temperature Transition Rate (OC/s)
AcquiSition Mode
• Melting Curve Analysis
40
Quantification
Segment I
95
0
20
None
Parameter
Value
Cycles
Type
Melting curve
Segment I
Target temperature [0C]
95
Incubation Time (s)
0
Temperature Transition Rate (O Cls)
20
Acquisition Mode
None
Fluorimeter gain of channel 1 was set to 1.
Segment 2
Segment 3
55
72
5
8
20
20
Single
one
Segment 2
Segment 3
65
95
30
0
20
0.1
None
Cont.