abi 7500 - Prepare the Sample Dilutions

Chapter 7 Prepare the Comparative C
T
Reactions
Prepare the Sample Dilutions
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
142
Notes
Prepare the Sample Dilutions
Perform sample dilutions before adding the samples to the final reaction mix. Dilute the
samples using the volumes that were calculated by the 7500 software (“Select the Sample
Dilution Calculations tab.” on page 128).
About the
Example
Experiment
For the comparative C
T
example experiment:
• Sample dilutions are necessary because the sample volume is limited to 10% of the
total reaction volume in the 7500 software. Because the total reaction volume is
50 µL/reaction, the sample volume is 5 µL/reaction.
• The stock sample concentration is 100 ng/µL. After diluting the sample according to
the Sample Dilutions Calculations table, the sample has a concentration of
50 ng/µL. Adding 5 µL at this concentration to the final reaction mix volume of
50 µL yields a 1✕ concentration in the final reaction.
• The volumes calculated in the software are:
Required
Materials
• Water (to dilute the sample)
• Microcentrifuge tubes
• Pipettors
• Pipette tips
• Sample stock
•Vortexer
• Centrifuge
Sample
Name
Stock Concentration
(ng/µL)
Sample
Volume (µL)
Diluent
Volume (µL)
Total Volume of
Diluted Sample (µL)
Brain 100.0 66.0 66.0 132.0
Kidney 100.0
66.0 66.0 132.0
Liver 100.0
66.0 66.0 132.0
Universal 100.0
66.0 66.0 132.0