Chapter 3
Prepare the Relative Standard Curve Reactions
Prepare the Reaction Plate
63
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
5.
Centrifuge the reaction plate briefly to remove air bubbles.
6.
Verify that the liquid is at the bottom of each well of the reaction plate. If not,
centrifuge the reaction plate again at a higher speed and for a longer period of time.
IMPORTANT!
Do not allow the bottom of the reaction plate to become dirty. Fluids
and other contaminants that adhere to the bottom of the reaction plate can
contaminate the sample block and cause an abnormally high background signal.
7.
Until you are ready to perform the run, place the reaction plate on ice in the dark.
Preparation
Guidelines
When you prepare your own relative standard curve experiment:
• Make sure you use the appropriate consumables.
• Make sure the arrangement of the PCR reactions matches the plate layout displayed
in the 7500 software. You can either:
– Accept the plate layout automatically generated by the software.
or
– Use Advanced Setup to change the plate layout in the software.
• If you use optical adhesive film, seal each reaction plate as follows:
a.
Place the reaction plate onto the center of the 96-well base.
b.
Load the reaction plate as desired.
c.
Remove a single optical adhesive film (film) from the box.
Fold back one of the end-tabs. Hold the film with its
backing side up.
d.
In one continuous movement, peel back the white
protective backing from the center sealing surface. Do not
touch the center sealing surface.
IMPORTANT!
Improper peeling of the optical adhesive film may result in
haziness, but does not affect results. Haziness disappears when the film comes
into contact with the heated cover in the instrument.
Correct
Incorrect
Liquid is at the
bottom of the well.
• Not centrifuged with enough force,
or
• Not centrifuged for enough time
