Chapter 3 Prepare the Relative Standard Curve Reactions
Prepare the Reaction Mix
59
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
Prepare the
Reaction Mix
IMPORTANT! Prepare the reaction mix for each target assay separately.
CHEMICAL HAZARD. TaqMan
®
Universal PCR Master Mix
may cause eye and skin irritation. Exposure may cause discomfort if swallowed or
inhaled. Read the MSDS, and follow the handling instructions. Wear appropriate
protective eyewear, clothing, and gloves.
1. Label an appropriately sized tube for each reaction mix:
• c-myc Reaction Mix
• GAPDH Reaction Mix
2. For the c-myc assay, add the required volumes of each component to the c-myc
Reaction Mix tube:
3. For the GAPDH assay, add the required volumes of each component to the GAPDH
Reaction Mix tube:
4. Mix the reaction mix in each tube by gently pipetting up and down, then cap each
tube.
5. Centrifuge the tubes briefly to remove air bubbles.
6. Place the reaction mixes on ice until you prepare the reaction plate.
Component
Volume (
µ
L)
for 1 Reaction
Volume (
µ
L) for 24
Reactions
Plus 10% Excess
Ta qM an
®
Universal PCR Master Mix (2
✕
)
25.0 660.0
c-myc Assay Mix (20
✕
)
2.5 66.0
Water
17.5 462.0
Total Reaction Mix Volume
45.0 1188.0
Component
Volume (
µ
L)
for 1 Reaction
Volume (
µ
L) for 24
Reactions
Plus 10% Excess
Ta qM an
®
Universal PCR Master Mix (2
✕
)
25.0 660.0
GAPDH Assay Mix (20
✕
)
2.5 66.0
Water
17.5 462.0
Total Reaction Mix Volume
45.0 1188.0
To Next Page
Loading...