Chapter 3 Prepare the Relative Standard Curve Reactions
Prepare the Reaction Plate
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
62
Notes
2. For each replicate group, prepare the standard reactions:
a. To appropriately sized tubes, add the volumes of reaction mix and standard
listed below.
b. Mix the reactions by gently pipetting up and down, then cap the tubes.
c. Centrifuge the tubes briefly to remove air bubbles.
d.
Add 50 µL of the standard reaction to the appropriate wells in the reaction plate.
3. For each replicate group, prepare the reactions for the unknowns:
a. To appropriately sized tubes, add the volumes of reaction mix and sample listed
below.
b. Mix the reactions by gently pipetting up and down, then cap the tubes.
c. Centrifuge the tubes briefly to remove air bubbles.
d. Add 50 µL of the unknown (sample) reaction to the appropriate wells in the
reaction plate.
4. Seal the reaction plate with optical adhesive film.
Tube Standard Reaction Reaction Mix Reaction Mix Volume (
µ
L) Standard
Standard Volume
(
µ
L)
1 c-myc Std 1 c-myc reaction mix 148.5 c-myc Std 1 16.5
2 c-myc Std 2 c-myc reaction mix 148.5 c-myc Std 2 16.5
3 c-myc Std 3 c-myc reaction mix 148.5 c-myc Std 3 16.5
4 c-myc Std 4 c-myc reaction mix 148.5 c-myc Std 4 16.5
5 c-myc Std 5 c-myc reaction mix 148.5 c-myc Std 5 16.5
6 GAPDH Std 1 GAPDH reaction mix 148.5 GAPDH Std 1 16.5
7 GAPDH Std 2 GAPDH reaction mix 148.5 GAPDH Std 2 16.5
8 GAPDH Std 3 GAPDH reaction mix 148.5 GAPDH Std 3 16.5
9 GAPDH Std 4 GAPDH reaction mix 148.5 GAPDH Std 4 16.5
10 GAPDH Std 5 GAPDH reaction mix 148.5 GAPDH Std 5 16.5
Tube Unknown Reaction Reaction Mix Reaction Mix Volume (
µ
L) Sample Sample Volume (
µ
L)
1 c-myc Liver c-myc reaction mix 148.5 Liver 16.5
2 c-myc Kidney c-myc reaction mix 148.5 Kidney 16.5
3 GAPDH Liver GAPDH reaction mix 148.5 Liver 16.5
4 GAPDH Kidney GAPDH reaction mix 148.5 Kidney 16.5
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