Chapter 3
Prepare the Relative Standard Curve Reactions
Prepare the Standard Dilution Series
57
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
2.
Add the required volume of water (diluent) to each empty tube:
3.
In the GAPDH Std. 1 tube:
a.
Vortex the stock for 3 to 5 seconds, then centrifuge the tube briefly.
b.
Using a new pipette tip, add 8.07 µL of stock to the GAPDH Std. 1 tube.
c.
Vortex Std. 1 for 3 to 5 seconds, then centrifuge the tube briefly.
4.
In the GAPDH Std. 2 tube:
a.
Using a new pipette tip, add 2.02 µL of dilution 1 to GAPDH Std. 2 tube.
b.
Vortex Std. 2 for 3 to 5 seconds, then centrifuge the tube briefly.
5.
In the GAPDH Std. 3 tube:
a.
Using a new pipette tip, add 2.02 µL of dilution 2 to the GAPDH Std. 3 tube.
b.
Vortex Std. 3 for 3 to 5 seconds, then centrifuge the tube briefly.
6.
In the GAPDH Std. 4 tube:
a.
Using a new pipette tip, add 2.02 µL of dilution 3 to the GAPDH Std. 4 tube.
b.
Vortex Std. 4 for 3 to 5 seconds, then centrifuge the tube briefly.
7.
In the GAPDH Std. 5 tube:
a.
Using a new pipette tip, add 2.02 µL of dilution 4 to the GAPDH Std. 5 tube.
b.
Vortex Std. 5 for 3 to 5 seconds, then centrifuge the tube briefly.
8.
Place the standards on ice until you prepare the reaction plate.
Preparation
Guidelines
When you prepare your own relative standard curve experiment:
• Standards are critical for accurate analysis of run data.
• Any mistakes or inaccuracies in making the dilutions directly affect the quality of
results.
• The quality of pipettors and tips and the care used in measuring and mixing
dilutions affect accuracy.
• Use TE buffer or water to dilute the standards.
Tube
Standard Name
Volume of Diluent to Add (µL)
1
GAPDH Std. 1
12.10
2
GAPDH Std. 2
18.15
3
GAPDH Std. 3
18.15
4
GAPDH Std. 4
18.15
5
GAPDH Std. 5
18.15
