Chapter 3 Prepare the Reactions
Prepare the Reaction Plate
53
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve
Experiments
Notes
3. For each replicate group, prepare the reactions for the unknowns:
a. To appropriately sized tubes, add the volumes of reaction mix and sample listed
below.
b. Mix the reactions by gently pipetting up and down, then cap the tubes.
c. Centrifuge the tubes briefly to remove air bubbles.
d. Add 25 µL of the unknown (sample) reaction to the appropriate wells in the
reaction plate.
4. Seal the reaction plate with optical adhesive film.
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. Place the reaction plate on ice in the dark until you are ready to perform the run.
Preparation
Guidelines
When you prepare your own 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.
Tube Unknown Reaction Reaction Mix
Reaction Mix
Volume (µL)
Sample
Sample Volume
(µL)
1 RNase P pop1 RNase P reaction mix 74.25 pop1 8.25
2 RNase P pop2 RNase P reaction mix 74.25 pop2 8.25
Correct Incorrect
Liquid is at the
bottom of the well.
• Not centrifuged with enough force, or
• Not centrifuged for enough time
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