Bio-Rad SsoAdvanced™ Universal SYBR® Green Supermix - Manual (Page 10)

SsoAdvanced ™ Universal SYBR ® Green Supermix Instruction Manual 4 |
Procedure
Reaction Mix Preparation and Thermal Cycling Protocol
1. Thaw SsoAdvanced ™ universal SYBR ® Green 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 SYBR ®
Green supermix (2x) 10 μl 5 μl 1x
Forward and reverse primers Variable Variable 250–500 nM each
Template (add at step 4) Variable Variable cDNA: 100 ng–100 fg
Genomic DNA: 50 ng–5 pg
Nuclease-free H
2
O Variable Variable —
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.