SsoFast™ EvaGreen® Supermix

QuickGuide
Sso7d-fusion Polymerase Converting from a SYBR Green mix to SsoFast
The unique antibody hot-start Sso7d-fusion polymerase in SsoFast EvaGreen
supermix enables fast cycling without compromising PCR sensitivity, efficiency,
and reproducibility. The dsDNA-binding protein, Sso7d (orange), stabilizes the
polymerase:template complex to deliver the performance advantages listed
below:
polymerase

Sso7d

Ease of protocol conversion from SYBR Green mix to SsoFast EvaGreen supermix.
Robust detection of the human
-tubulin gene target from cDNA using optimized
conditions (60°C annealing/extension) for a SYBR Green mix (left panel). A 2-step gradient
55°C (blue traces) to 67°C (red traces) cycling protocol (5s combined annealing/extension)
was used to determine optimal conditions for SsoFast EvaGreen supemix (right panel).
SYBR Green Ct (60°C) = 23.5; SsoFast EvaGreen average Ct (57°C to 60°C) = 22.5.

Optimizing your cycling protocol

Tolerance to many common PCR inhibitors provides increased
sensitivity and better reproducibility.

Improved speed and processivity results in reduced overall

reaction times.

EvaGreen Dye
EvaGreen dye is a fluorescent nucleic acid binding dye with spectral properties
similar to SYBR® Green I and fluorescein. Unlike SYBR Green I, EvaGreen dye
exhibits very low PCR inhibition, making it an ideal choice for fast qPCR
protocols. It can be used at a higher concentrations to deliver the following
performance advantages:
SsoFast EvaGreen supermix delivers the best results using an optimized 2-step

Next generation, saturating dye generates greater fluorescence cycling protocol. Detection of the human 18S rRNA gene target from cDNA using a
standard 3-step cycling protocol (15s annealing + 30s extension) results in undesirable
and increased sensitivity. amplification during the later cycles (purple circles - left panel). A fast 3-step cycling protocol
(5s annealing + 5s extension) shows significant improvement (purple circles - middle panel).

Ideal for fast qPCR protocols and high resolution melting (HRM However, the best results (right panel) are generated using an optimized 2-step cycling
analysis). protocol (combined 5s annealing/extension).

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 SsoFast™ EvaGreen® Supermix
QuickGuide
Recommendations for Optimal Results Optimized Cycling Conditions for qPCR
For existing primers, use a 2-step (gradient) cycling protocol Bio-Rad CFX96™ and CFX384™ Real-Time PCR Systems
with 55°C to 60°C annealing/extension temperatures. This Cycling Step
cDNA or Plasmid DNA
Temperature Time # Cycles
Genomic DNA
Temperature Time
should provide good results for a broad range of primers. Enzyme activation 95°C 30 sec 1 98°C 2 min
Denaturation 95°C 1–5 sec 98°C 1–5 sec
30–40
Annealing/Extension 55–60°C 1–5 sec 55–60°C 1–5 sec
For new primers, use the Primer3 (http://frodo.wi.mit.edu/) Melt curve
65–95°C
(in 0.5°C inc.)
2–5 sec/step 1
65–95°C
(in 0.5°C inc.)
2–5 sec/step

program under default settings to design primers with melting

temperatures of 60°C, and use this as the annealing Bio-Rad iQ, iQ5, MyiQ, and MyiQ2 Real-Time PCR Systems
temperature. cDNA or Plasmid DNA Genomic DNA
Cycling Step Temperature Time # Cycles Temperature Time
Enzyme activation 95°C 30 sec 1 98°C 2 min
Avoid using annealing/extension temperatures above 60°C. Denaturation 95°C 5 sec 98°C 5 sec
30–40
Annealing/Extension 55–60°C 10 sec 55–60°C 10 sec
65–95°C 65–95°C
The recommended optimal primer concentration is 500 nM, Melt curve
(in 0.5°C inc.)
10 sec/step 1
(in 0.5°C inc.)
10 sec/step

however 300 nM primer concentrations can also be used.

Roche LightCycler® 480 Real-Time PCR System
Suggested input quantities of template are: cDNA or Plasmid DNA Genomic DNA
Cycling Step Temperature Time # Cycles Temperature Time
- cDNA from 50 ng to 50 fg of total RNA Enzyme activation 95°C 30 sec 1 98°C 2 min
Denaturation 95°C 5 sec 98°C 5 sec
- genomic DNA: 50 ng to 5 pg Annealing/Extension 55–60°C 20 sec
30–40
55–60°C 20 sec
Melt curve 65–95°C continuous 1 65–95°C continuous

Applied Biosystems 7500/7500 Fast Real-Time PCR Systems

cDNA or Plasmid DNA Genomic DNA
General Considerations Cycling Step Temperature Time # Cycles Temperature Time
Enzyme activation 95°C 30 sec 1 98°C 2 min
Denaturation 95°C 5 sec 98°C 5 sec
Full activation of the hot-start Sso7d-fusion polymerase occurs Annealing/Extension 55–60°C 15–30 sec
30–40
55–60°C 15–30 sec
Melt curve 65–95°C continuous 1 65–95°C continuous
within 30 seconds at 95°C. Note: ROX passive reference dye (catalog #172-5858) must be added to a final concentration of 0.075x for use on this

EvaGreen dye exhibits sufficient background fluorescence to Licensing Statements

perform dynamic well factor collection for data normalization on EvaGreen is a trademark of Biotium Inc. Bio-Rad Laboratories, Inc. is licensed by Biotium Inc. to sell reagents containing EvaGreen dye for use in
real-time PCR, for research purposes only.

the iQ™, iQ™5, MyiQ™, or MyiQ™2 Real-Time PCR systems. Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the
purchaser’s own internal research. No other patent rights are conveyed expressly, by implication, or by estoppel. Further information on
purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California

Longer initial denaturation times (up to 3 min) & higher

84404, USA.

HRM is a trademark of Corbett Life Sciences and its subsidiaries. SYBR is a trademark of Invitrogen Corporation.
temperatures (up to 98°C) may be required for complete Bio-Rad’s real-time thermal cyclers are licensed real-time thermal cyclers under Applera’s United States Patent 6,814,934 B1 for use in research

denaturation of genomic DNA.

and for all other fields except the fields of human diagnostics and veterinary diagnostics.

This product is covered by one or more of the following U.S. patents or their foreign counterparts owned by Eppendorf AG: U.S. Patents 6,767,512
and 7,074,367.

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