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
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