Chapter 7 Prepare the Comparative C
T
Reactions
Prepare the Sample Dilutions
143
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
Prepare the
Sample Dilutions
1. Label a separate microcentrifuge tube for each diluted sample:
•Brain
• Kidney
•Liver
•Universal
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 comparative C
T
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)
1Brain 66.0
2 Kidney 66.0
3Liver 66.0
4Universal 66.0
Tube Sample Name Sample Volume (µL)
1 Brain 66.0
2 Kidney 66.0
3 Liver 66.0
4 Universal 66.0
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