Quantification of Human Genomic DNA Using Retinoic X Receptor B Gene •
Preparation of SYBR Green I master mix and hybridization probes master mix:
Volume (fill
IFinal]
SYBR Green I Hybridization
H2O
10.4
8.4
MgCl2 (25 mMII)
1.6
1.6
3mM
Primers (10 f1M each)
1+1
1+1
0.5 flM each
UghtCyder FastStart DNA Master
2
Ix
SYBR Green I
UghtCyder Hybridization
2
Ix
Probe Master
Hybridization probes (3 f1M each)
1+1
0.15 flM each
Total master mix volume per
16
16
20 f.ll final volume
reaction
For n peR reactions, reagents for n+ 1 reactions were mixed and 16 ~ pipetted
into each precooled capillary. To reduce pipetting error, 4 III of standard or test
DNA template were then added.
Protocol for amplification with SYBR Green I:
Parameter
Program I
Program 2
Program 3
.
Cycles
40
:rYP e
Denaturation
Quantification
Melting Curve
Seg 1
Seg 1 Seg2 Seg 3
Seg I
Seg 2 Seg 3
Target temperature
roC)
95
95
60
72
95
70
40
Incubation time [sl
600
15
5
10
30
0
50
Temperature
transition rate [O C/s ]
20
20
20
20
20
0.1
20
Acquisition mode
None
one None Single
Cont
Cont
Cont
Gains
Fl=4
FI/l
Protocol for amplification with hybridization probes:
Parameter
Program I
Program 2
Program 3
Cycles
40
I
Type
Denaturation
Quantification
Cooling
Seg 1
Seg 1 Seg 2 Seg3
Seg 1
Target temperature roC)
9S
9S
60
72
40
Incubation time [sl
600
15
10
10
30
Temperature transition rate [OC/s1 20
20
20
5
20
Acquisition mode
None
None Single None
None
Gains
Fl=5; F2=15