abi 7500 - Prepare the Standard Dilution Series

Chapter 3 Prepare the Relative Standard Curve Reactions
Prepare the Standard Dilution Series
55
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
Prepare the Standard Dilution Series
You prepare the standard dilution series using the volumes that were calculated by the 7500
software (“To complete the Reaction Mix Calculations tab for the c-myc assay” on page 37
and “To complete the Reaction Mix Calculations tab for the GAPDH assay” on page 38):
About the
Example
Experiment
For the relative standard curve example experiment:
• The standard (stock) concentration is 100 ng/µL.
• The volumes calculated in the software for both the c-myc and GAPDH assays are:
Required
Materials
• Water (to dilute the standards)
• Microcentrifuge tubes
• Pipettors
• Pipette tips
• Standard stock
•Vortexer
• Centrifuge
Prepare the
Standard Dilution
Series for the
c-myc Assay
1. Label a separate microcentrifuge tube for each standard:
• c-myc Std. 1
• c-myc Std. 2
• c-myc Std. 3
• c-myc Std. 4
• c-myc Std. 5
Dilution Point
(Factor)
Source
Source Volume
(µL)
Diluent Volume
(µL)
Total Volume
(µL)
Standard Concentration
(ng/µL)
1 (200) Stock 8.07 12.10 20.17 40.0
2 (20) Dilution 1 2.02 18.15 20.17 4.0
3 (2) Dilution 2 2.02 18.15 20.17 0.4
4 (0.2) Dilution 3 2.02 18.15 20.17 0.04
5 (0.02) Dilution 4 2.02 18.15 20.17 0.004