Bio-Rad SsoAdvanced™ Universal Probes Supermix - Manual (Page 12)

SsoAdvanced ™ Universal Probes Supermix Instruction Manual 6 |
Tips to Get Started
■ ■
Always evaluate the performance of the supermix following the recommended reaction and
cycling conditions prior to modification
■ ■
Be sure to set the activation time to 30 sec for cDNA and 2–3 min for genomic DNA
■ ■
The 2x supermix has been optimized for 20 µl reactions in 96-well plates and 10 µl reactions
in 384-well plates
Procedure
Reaction Mix Preparation and Thermal Cycling Protocol
1. Thaw SsoAdvanced ™ universal probes supermix and other frozen reaction components to
room temperature. Mix thoroughly, centrifuge briefly to collect solutions at the bottom of
tubes, and then store on ice protected from light.
2. Prepare (on ice or at room temperature) enough reaction setup for all qPCR reactions
by adding all required components except the template according to the following
recommendations (Table 1).
Table 1. Reaction setup.*
Volume per Volume per
Component 20 μl Reaction 10 μl Reaction Final Concentration
SsoAdvanced universal
probes supermix (2x)
10 μl 5 μl 1x
Total reaction mix volume 20 μl 10 μl —
* Scale all components proportionally according to sample number and reaction volumes.
3. Mix the assay master mix thoroughly to ensure homogeneity and dispense equal aliquots
into each PCR tube or into the wells of a PCR plate. Good pipetting practice must be
employed to ensure assay precision and accuracy.
4. Add samples (and nuclease-free H
2
O if needed) to the PCR tubes or wells containing the
reaction setup (Table 1), seal tubes or wells with flat caps or optically transparent film, and
vortex 30 sec or more to ensure thorough mixing of the reaction components. Spin the tubes
or plate to remove any air bubbles and collect the reaction mixture in the vessel bottom.
5. Program thermal cycling protocol on the real-time PCR instrument according to Table 2.
6. Load the PCR tubes or plate onto the real-time PCR instrument and start the PCR run.
7. Perform data analysis according to the instrument-specific instructions.
Forward and reverse primers Variable Variable 250–900 nM each
Fluorogenic probe Variable Variable 150–250 nM each
Template (add at step 4) Variable Variable cDNA: 100 ng–100 fg
Genomic DNA: 5 00 ng–5
pg Nuclease-free H
2
O Variable Variable —