ThermaStop™

 

How do I re-suspend ThermaStop for use?

 

To prepare 5 Units/µl ThermaStop, add 100 µl 10 mM molecular-grade Tris-Cl, pH 8.3 to 500 units dry reagent (500 µl 10 mM Tris-Cl, pH 8.3 to 2500 units dry reagent), vortex 1-2 minutes, then centrifuge briefly. Allow tube to sit at room temperature for 15 minutes with occasional mixing to ensure reagent is completely dissolved.

 

Do I need to centrifuge the material after re-suspension?

 

Yes, centrifuge briefly to bring any liquid to bottom of the tube.

 

How long can I store the dry material?

 

Dried ThermaStop can be stored for at least a year if kept at room temperature in the vial provided.

 

How long can I store the liquid material?

 

Liquid ThermaStop can be stored for up to six months if kept at 4°C or -20°C.

 

What polymerase enzymes work with ThermaStop?

 

ThermaStop is compatible with most DNA-dependent DNA polymerases, including all native and recombinant Taq DNA polymerases, heat-activated Taq DNA polymerases, and Tfi DNA polymerase.  ThermaStop has been tested with the following commercially available enzymes:

 

Non-hot start polymerase enzymes;

a) Invitrogen Recombinant Taq DNA Polymerase

b) Promega Go Taq® DNA Polymerase

c) Syd Labs Taq DNA Polymerase

d) Hain Life Sciences Taq DNA Polymerase

 

Hot start polymerase enzymes;

a) Platinum™ Taq DNA Polymerase

b) Promega Go Taq® DNA Polymerase

c) Applied Biosystems™  AmpliTaq Gold™ DNA Polymerase

d) LGC KlearTaq™ DNA Polymerase

 

Can I add ThermaStop directly to my Master Mix?

 

Yes. It is not necessary to pre-incubate a DNA polymerase with ThermaStop. ThermaStop should be added to the master mix  before adding the primers and DNA targets.

 

How long can I wait after adding ThermaStop before starting my reaction?

 

Once ThermaStop is added to a PCR master mix the reaction can be started right away.  If desired, a PCR master mix with ThermaStop can be stored for up to 24 hours at room temperature prior to PCR.

 

How many units of ThermaStop do I use with my Taq Polymerase?

 

Add an equal number of units of ThermaStop and Taq DNA polymerase to a PCR master mix. For reactions producing large amounts of non-specific products, add up to 1.5-4 units ThermaStop per unit of Taq DNA polymerase.

 

Can I use ThermaStop in an RT-PCR assay?

 

We recommend ThermaStop-RT for all RT-PCR assays.

 

Can I use ThermaStop in a Multiplex PCR assay?

 

Yes. We also recommend using ThermaStop and ThermaGo, a reagent that enhances the specificity of Taq DNA polymerase by suppressing mis-priming during the annealing and extension steps of PCR.

 

What kind of improvement should I see with ThermaStop?

 

ThermaStop will reduce the production of primer dimers and non-specific products and generate higher yields of the correct products (e.g. stronger probe signals, correct bands on gels) compared to non-hot start conditions.

 

 

 

ThermaGo™

 

How do I re-suspend ThermaGo for use?

 

To prepare 5 Units/µl ThermaStop, add 100 µl 10 mM molecular-grade Tris-Cl, pH 8.3 to 500 units dry reagent (500 µl 10 mM Tris-Cl, pH 8.3 to 2500 units dry reagent), vortex 1-2 minutes, then centrifuge briefly. Allow tube to sit at room temperature for 15 minutes with occasional mixing to ensure reagent is completely dissolved.

 

Do I need to centrifuge the material after re-suspension?

 

Yes, centrifuge briefly to bring any liquid to bottom of the tube.

 

How long can I store the dry material?

 

Dried ThermaGo can be stored for at least a year if kept at room temperature in the vial provided.

 

How long can I store the liquid material?

 

Liquid ThermaStop can be stored for up to six months if kept at 4°C.

 

What polymerase enzymes work with ThermaGo?

 

ThermaGo is compatible with all Taq DNA polymerases.

 

Can I add ThermaGo directly to my Master Mix?

 

Yes. It is not necessary to incubate a PCR DNA polymerase with ThermaGo.

 

How long can I wait after adding ThermaGo before starting my reaction?

 

Once ThermaGo is added to a PCR master mix the reaction can be started right away.

 

How many units of ThermaGo do I use with my Taq Polymerase?

 

To use, begin by adding an equal number of units of ThermaGo and Taq DNA polymerase to a PCR master mix. To further optimize, test ThermaGo in the range of 0.5 to ≥2.0 Units per Unit of Taq DNA polymerase. Note: very high levels of ThermaGo can inhibit amplification.

 

Can I use ThermaGo in a Multiplex PCR assay?

 

Yes. We also recommend using ThermaGo together with ThermaStop, a reversible hot start reagent compatible with all Taq DNA polymerases. ThermaStop 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.

 

What kind of improvement should I see with ThermaGo?

 

Reactions containing optimal levels of ThermaGo will exhibit greater quantitative reproducibility due to suppression of non-specific products. Use of ThermaGo will produce higher yields of the intended products (e.g. stronger probe signals, correct bands on gels).

 

 

 

 

ThermaStop-RT™

 

How do I re-suspend ThermaStop-RT for use?

 

To prepare a stock solution of 5 Units/µl ThermaStop-RT, add 50 µl 10 mM molecular-grade Tris-Cl, pH 8 to 250 units dry reagent (250 µl 10 mM Tris-Cl, pH 8 to 1250 units dry reagent), vortex 1-2 minutes, then centrifuge briefly. Allow tube to sit at room temperature for 15 minutes with occasional mixing to ensure reagent is completely dissolved.

 

How long can I store the dry material?

 

Dried ThermaStop-RT can be stored for at least a year if kept at room temperature in the vial provided.

 

How long can I store the liquid material?

 

Liquid ThermaStop-RT can be stored for at least six months at 4°C or -20°C.

 

What reverse transcripases work with ThermaStop-RT?

 

ThermaStop-RT should be compatible with all MMLV-derived reverse transcriptase, although best results are obtained with those which can be used at 50oC or higher.  ThermaStop-RT has been tested with SuperScript III (Invitrogen), PrimeScript (Takara), qScript (QuantaBio), and tests with additional enzymes are ongoing.  Contact ThermaGenix technical support if you have questions regarding use with a specific enzyme.

 

Can reverse transcription be done at temperatures below 50oC with ThermaStop-RT?

 

The activity of reverse transcriptase is partially inhibited by ThermaStop-RT at 45oC, but it may be possible to get good results at that temperature if the duration of the step is increased.  However, higher temperatures improve primer specificity and temperatures of 55oC or higher are recommended for best results, if the enzyme is stable at that temperature.

 

What types of primers can be used with ThermaStop-RT?

 

ThermaStop-RT works with gene-specific primers.  Primers designed with computer programs can typically be used at concentrations of 0.5 micromolar or higher without generating detectable non-specific amplification.

 

Can ThermaStop-RT be used with random primers or oligo(dT)?

 

We do not recommend the use of random primers or typical oligo(dT) due to the need for reverse transcriptase activity at low temperatures.  It may be possible to use longer oligo(dT)20 at 50oC, but this has not been tested.

 

How much ThermaStop-RT should be used?

 

One Unit of ThermaStop-RT is usually sufficient in a 20 µl reaction containing 50 Units of reverse transcriptase (e.g. SuperScript III or PrimeScript).

 

Can ThermaStop-RT be added to One-Step RT-PCR kits?

 

Yes.  Add ThermaStop-RT before adding primers and RNA. It may be necessary to test different concentrations of ThermaStop-RT with one-step RT-PCR kits containing unknown enzyme concentrations.  Contact ThermaGenix technical support for more information about use of ThermaStop-RT with a specific kit.

 

Can I prepare my reagent mixture at ambient temperature if I add ThermaStop-RT?

 

Athough ThermaStop-RT greatly reduces reverse transcriptase activity at room temperature, a low-level activity remains.  We recommend that master mixes with ThermaStop-RT are mixed with primers and RNA while on ice to obtain best results.

 

Can I use ThermaStop in an RT-PCR assay containing ThermaStop-RT?

 

We recommend Taq antibodies or a heat-activated polymerase be used to keep the DNA polymerase inactive during the RT incubation of a one-step RT-PCR.

 

Can I use ThermaGo in an RT-PCR assay containing ThermaStop-RT?

 

We do not recommend using ThermaGo in one-step RT-PCR.  ThermaGo can be used for PCR of cDNA that is purified or highly diluted following the reverse transcription step of two-step RT-PCR.

 

Can I use ThermaStop-RT in a Multiplex RT-PCR assay?

 

Yes.  ThermaStop-RT enables accurate multiplexing by preventing mis-priming that is likely with high numbers of primer pairs.

 

What kind of improvement should I see with ThermaStop-RT?

 

ThermaStop-RT will reduce non-specific amplification in RT-PCR using gene-specific primers, and thereby improve amplification rates and final yield of specific products.  Greatest improvement is obtained for samples with low concentrations of RNA; assay sensitivity may be improved 10-fold or more.  Non-specific products should not be detected using post-PCR melts with SYBR Green or using gel electrophoresis.  The high specific product yield and absence of non-specific products should also provide better results for sequencing and other analysis methods.