ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

Programming
Methods for Allelic
Discrimination
Programming the
Method for Absolute
Quantification
4-14 Run Setup and Basic Operation
The SDS software is designed to maximize instrument throughput and therefore does
not provide the option to thermal cycle allelic discrimination plate documents.
Because allelic discrimination experiments are end-point runs that do not require data
collection during the PCR, thermal cycling can be performed on a dedicated thermal
cycler and then transferred to the 7900HT instrument for data collection and analysis.
If you want to thermal cycle the
allelic discrimination plates on… Then…
a designated thermal cycler go on to “Step 5 – Saving the Plate Document as a
Template” on page 4-17
the 7900HT instrument follow the procedure below.
Performing Thermal Cycling of Allelic Discrimination Plates on the 7900HT Instrument
To perform the thermal cycling and the plate read using the 7900HT instrument, run
the plate first as a real-time plate document and then again as an allelic discrimination
plate document as explained below.
IMPORTANT Follow the procedure below only if you intend to perform the PCR on the
7900HT instrument. Otherwise, perform the PCR on a dedicated thermal cycler and then
transfer the plate to the 7900HT instrument for data collection.
To conduct allelic discrimination thermal cycling on the 7900HT instrument:
Step Action
1 Launch the SDS software.
2 Create a real-time plate document for absolute quantification as described on
page 4-6.
Note It is not necessary to configure the plate document with detectors.
3 Program the plate document method with the thermal cycling times and
temperatures for your protocol as described in “Programming the Method for
Absolute Quantification,” below.
4 Run the plate using the real-time plate document as described on page 4-21.
Note Although large, the real-time file may be helpful in diagnosing and
troubleshooting the experiment later if the data from the allelic discrimination run
produces unexpected results.
5 Go on to “Step 5 – Saving the Plate Document as a Template” on page 4-17.
Note The following procedure describes how to configure only the basic features of the
method: thermal cycler conditions, sample volume, and data collection options. To further
customize the method for the plate document, click the button in the Instrument tab and refer
to the online help for instructions on configuring the auto increment and ramp rate values.
To create a method for the absolute quantification run:
Step Action
1 From the SDS software, click the Instrument tab of the plate document.
2 If necessary, select or de-select the 9600 Emulation check box.
Note When the 9600 Emulation check box is checked, the SDS Software reduces
the ramp rate of the 7900HT instrument to match that of the ABI PRISM® 7700
Sequence Detection System instrument.