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

Assigning Quantities
to Standards for
Absolute
Quantification
Setting the
Passive Reference
4-12 Run Setup and Basic Operation
For the SDS software to create a standard curve for quantification of unknown
samples, absolute quantification plate documents must contain quantity values for the
standards contained on the plate. The software expresses quantity as the number of
copies of the target sequence present within an individual well of the plate.
IMPORTANT The SDS software excludes from analysis data from Standard wells assigned a
Quantity value of 0.
To apply quantities to the plate document:
Step Action
1 From the plate grid of the SDS software, select the replicate wells containing the
first standard in the dilution series.
2 From the well inspector, click the text field in the Quantity column for the appropriate
detector, type the number of copies of the target template the replicate wells
contain, and press Enter.
The software labels the selected standard wells with the specified quantity.
Quantity
text field
3 Repeat steps 1 and 2 to configure the other sets of replicate standard wells on the
plate with the appropriate quantities.
When finished, the plate grid should contain a complete set of replicate wells
labeled with the Standard task and assigned quantities which the software will use
to compute the standard curve for the run.
4 If necessary, set the passive reference for the plate document as explained below.
Otherwise, program the method for your run as explained on page 4-13.
If using an Applied Biosystems chemistry, use the default Passive Reference setting
(Applied Biosystems chemistries use the ROX passive reference molecule). If
running a custom chemistry, select a dye to use as a passive reference for the run.
Note Applied Biosystems recommends using a passive reference to normalize the signals
from the reporter dyes (see page A-6 for more information).
To set the passive reference dye for the plate document:
Step Action
1 From the Passive Reference drop-down list, select the appropriate reference dye.
Passive Reference
drop-down list
2 Program the method for the run as explained on page 4-13.
Select the appropriate
passive reference