8
Perform an experiment
Applied Biosystems ViiA
™
7 Real-Time PCR System Quick Reference Guide
Prepare the reactions
IMPORTANT! Wear powder-free gloves when you handle the plate or array card.
Prepare the plate
Guidelines for preparing the plate:
• Include excess volume in your calculations to provide for loss during reagent transfers.
• Use TE buffer or water to dilute the standards and samples.
• Prepare the reagents according to the manufacturer’s instructions.
• Keep the dilutions and assay mix protected from light, in the freezer, until you are ready to use it. Excessive exposure to light may
affect the fluorescent probes or dyes.
• To prepare the reactions:
– Mix the master mix thoroughly by swirling the bottle.
– Resuspend the assay mix by vortexing, then centrifuge the tube briefly.
– Thaw any frozen samples, resuspend them by vortexing, then centrifuge the tubes briefly.
• Verify that the liquid is at the bottom of each well of the plate. If not, centrifuge the plate again at a greater rpm and for a longer time.
IMPORTANT! Do not allow the bottom of the plate to become dirty. Fluids and other contaminants that adhere to the bottom of the
plate can contaminate the sample block(s) and cause an abnormally high background signal.
• (Genotyping) Prepare the reactions for each SNP separately
• Place the reaction plate or array card at 4 °C in the dark until you are ready to load it into the instrument.
Correct Incorrect
Liquid is at
bottom of well
• Not centrifuged with enough force, or
• Not centrifuged for enough time
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