Prepare The Sample Dilutions - Applied Biosystems 7500 Getting Started

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
Sample Dilution Calculations Tab" on page
About the
For the standard curve example experiment:
Example
Experiment
Sample Stock Concentration
Name (ng/µL)
pop1 100.0
pop2 100.0
Required
Materials
Prepare the
Sample Dilutions
Notes
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve
Experiments
• 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
25 µL/reaction, the sample volume is 2.5 µL/reaction.
• The stock concentration is 100 ng/µL. After diluting the sample according to the
Sample Dilutions Calculations table, the sample has a concentration of 6.6 ng/µL.
Adding 2.5 µL at this concentration to the final reaction mix volume of 25 µL yields
a 1✕ concentration in the final reaction.
• The volumes calculated in the software are:
Sample Volume
(µL)
1.0
1.0
• Water (to dilute the sample)
• Microcentrifuge tubes
• Pipettors
• Pipette tips
• Sample stock
• Vortexer
• Centrifuge
1. Label a separate microcentrifuge tube for each diluted sample:
• Population 1
• Population 2
2. Add the required volume of water (diluent) to each empty tube:
Tube Sample Name
1 Population 1
2 Population 2
Chapter 3 Prepare the Reactions

Prepare the Sample Dilutions

34).
Diluent Volume Total Volume of Diluted Sample
(µL)
14.15
14.15
Diluent Volume (µL)
14.15
14.15
("Complete the
(µL)
15.15
15.15
45