Prepare The Sample Dilutions - abi 7500 Getting Started Manual

Preparation When you prepare your own relative standard curve experiment, Applied Biosystems
Guidelines
recommends:
• That you first extract DNA or RNA from the tissue or sample.
• The following templates:
For More For more information on:
Information
• Preparing cDNA templates – Refer to the Applied Biosystems High-Capacity cDNA
• Preparing RNA or gDNA templates – Refer to the protocol for the purification

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 ("To complete the
Sample Dilution Calculations tab:" on page 40).
About the
For the relative standard curve example experiment:
Example
• Sample dilutions are necessary because the sample volume is limited to 10% of the
Experiment
• The stock sample concentration is 100 ng/µL. After diluting the sample according to
• The volumes calculated in the software are:
Notes
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C Experiments
T
Chapter 3 Prepare the Relative Standard Curve Reactions
– Complementary DNA (cDNA) – cDNA reverse-transcribed from total RNA
samples using a High-Capacity cDNA Reverse Transcription Kit.
– RNA – Purified total RNA or mRNA extracted from tissue or sample.
– Genomic DNA (gDNA) – Purified gDNA already extracted from tissue or
sample.
Reverse Transcription Kits Protocol. The protocol is not shipped with the High-
Capacity cDNA Reverse Transcription Kits. You can download the protocol from:
http://docs.appliedbiosystems.com/search.taf
reagents that you select. To locate Applied Biosystems purification reagents, visit:
http://www.appliedbiosystems.com/
total reaction volume in the 7500 software. Because the total reaction volume is
50 µL/reaction, the sample volume is 5 µL/reaction.
the Sample Dilutions Calculations table, the sample has a concentration of
50 ng/µL. Adding 17 µL at this concentration to the final reaction mix volume of
34 µL yields a 1✕ concentration in the final reaction.
Stock
Sample Name Concentration
(ng/µL)
Liver 100.0
Kidney 100.0

Prepare the Sample Dilutions

Sample Diluent
Volume (µL) Volume (µL)
17.0 17.0
17.0 17.0
Total Volume
of Diluted
Sample (µL)
34.0
34.0
53