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High-Precision PCR

ThermaStop

A Universal Reagent

 

ThermaStop maximizes PCR performance, improves polymerase specificity, enhances end quantitative data and achieves superior amplicon yield for downstream applications from genotyping to Gibson assembly, next generation sequencing sample preparation to diagnostics.

 

ThermaStop is a reversible hot start reagent that is compatible with all Taq DNA polymerases. It acts directly on the polymerase to prevent non-specific enzymatic activity below 50°C. Polymerase activity is fully restored at 60°C, but is inhibited again by ThermaStop by cooling the reaction.

 

ThermaStop:

 

1. Accomplishes superior amplicon yield with your polymerase.

 

2. Effectively suppresses formation of primer dimers/non-specific products before PCR.

 

3. Improves polymerase specificity and enhances end quantitative data.

 

4. Eliminates amplification in no-template controls.

 

5. Rapidly inactivated in first PCR cycle so no need for extended high temperature incubation.

 

6. Immediately re-activated upon cooling enabling resumption of PCR cycling & safeguards product integrity at endpoint for downstream applications.

 

7. Stable non-aptamer, chemically modified nucleic acid not affected by proteases

 

Applications of Interest:

 

Hot-start protection for all forms of PCR.

 

Enhanced detection of low-copy number targets (single-cell PCR, digital PCR, rare target detection in liquid biopies/tumor samples).

 

Enhances accuracy of highly multiplexed-PCR for targeted next-generation sequencing and other applications.

 

 

 

 

Universal Hot Start Reagent

ThermaStop Improves Product Yield and Sensitivity

Documents

Documents Manuals and Protocols

 

Material safety data sheets (MSDS)

 

ThermaStop Improves Hot Start Taq Polymerases

lane M; Jump Start Taq (Sigma)

lane 1; MyTaq HS (Bioline)

lane 2; Platinum Taq (Invitrogen)

lane 3: AccuStart II Taq (Quantabio)

lane 4; FastStart Taq (Roche)

lane 5; E-Gel® Low-Range DNA ladder

lane 6; Jump Start Taq (Sigma) with ThermaStop

lane 1; MyTaq HS (Bioline) with ThermaStop

lane 2; Platinum Taq (Invitrogen) with ThermaStop

lane 3: AccuStart II Taq (Quantabio) with ThermaStop

lane 4; FastStart Taq (Roche) with ThermaStop.

 

REACTION CONDITIONS:

A total reaction volume of 30 µl contained 1 × PCR buffer, MgCl2, dNTPs, hot-start polymerase, and primer set 2F/2R was used, at the concentrations recommended by each manufacturer. Either no ThermaStop or the equivalent units of ThermaStop to Taq units per reaction.  Cycling conditions were 95°C, 2 min; 10 cycles at 95°C, 15 sec; 65°C, 15 sec; 72°C, 45 sec, 25 cycles at 95°C, 15 sec; 60°C, 15 sec; 72°C, 45 sec, then extension at 72°C for 5 min.  PCR products (20µl) were visualized on a E-Gel™ EX 2% agarose gel.

 

Description;

A stringent test of polymerase activity prior to thermal activation1.  A simple test with two primers that have a three base pair overlap at their 3’ ends.  These primers form a primer dimer (see arrow above) if any polymerization occurs prior to the thermal cycling.

 

Results:

Four of the five hot start Taq polymerases showed primer dimer formation. Yet when ThermaStop was added, all polymerases showed no primer dimer formation.

 

1Stevens et al., Biotechinques 2016, 61(6):293-296.