Chapter 3 Prepare the Relative Standard Curve 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
54
Notes
Required
Materials
• Water (to dilute the sample)
• Microcentrifuge tubes
• Pipettors
• Pipette tips
• Sample stock
•Vortexer
• Centrifuge
Prepare the
Sample Dilutions
1. Label a separate microcentrifuge tube for each diluted sample:
•Liver
• Kidney
2. Add the required volume of water (diluent) to each empty tube:
3. Add the required volume of sample stock to each tube:
4. Vortex each diluted sample for 3 to 5 seconds, then centrifuge the tubes briefly.
5. Place the diluted samples on ice until you prepare the reaction plate.
Preparation
Guidelines
When you prepare your own relative standard curve experiment:
• Sample dilutions may be necessary because the sample volume is limited to 10% of
the total reaction volume in the 7500 software. You must perform the sample
dilutions before adding the samples to the final reaction mix.
• For optimal performance of TaqMan
®
Gene Expression Assays or Custom TaqMan
®
Gene Expression Assays, use 10 to 100 ng of cDNA template per 20-µL reaction.
• Use TE buffer or water to dilute the sample.
For More
Information
For more information on Applied Biosystems assays, refer to the:
• TaqMan
®
Gene Expression Assays Protocol
• Custom TaqMan
®
Gene Expression Assays Protocol.
Tube Sample Name
Diluent Volume
(µL)
1Liver 17.0
2 Kidney 17.0
Tube Sample Name
Sample Volume
(µL)
1Liver 17.0
2 Kidney 17.0
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