HotStart DNA Polymerase (PCR) 1000 U Product Description HotStart Taq DNA Polymerase is a chemically modified Taq DNA Polymerase whose
enzyme activities can only be activated after 3-5 minutes of incubation at 94°C. This
enzyme thus exhibits no polymerase activities before the onset of thermal cycling,
preventing non-specific DNA amplification and primer dimer formation. PCR products,
amplified up to 6 kb in length with HotStart Taq DNA Polymerase, contain a single base
(A) 3’ overhang.
| Product Components | 250 U | 1000 U |
| HotStart Taq DNA Polymerase (5 U/µl) | 50 µl | 200 µl |
| 10X PCR buffer, with Mg2+ | 1 ml | 3 ml |
| 25 mM MgSO4 | 1 ml | 1 ml |
Storage Buffer Components 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 50 % glycerol and 1.0 %
Triton®X-100.
Unit Definition One unit of enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides
into a polynucleotide fraction in 30 mins at 70°C.
Shipping and Storage Upon arrival, HotStart Taq DNA Polymerase should be stored at -20°C. Avoid repeated
freeze-thaw cycles of all HotStart Taq components to retain maximum performance.
All HotStart Taq components are stable for 1 year from the date of shipping if stored
and handled properly.
Protocol The following basic protocol serves as a general guideline and starting point for any
PCR amplification. Optimal reaction conditions (incubation times, temperatures,
concentration of Taq DNA Polymerase, primers, MgSO4 and template DNA) may vary
and need to be optimized for each specific PCR.
All PCR experiments should be assembled in a nuclease-free environment. In addition,
DNA sample preparation, reaction set-up and subsequent reaction(s) should be
performed in separate areas to avoid cross contamination.
A negative control reaction (omitting template DNA) should always be performed in
tandem with sample PCR to confirm the absence of DNA contamination.
1. Add the following components to a sterile 0.2 ml PCR tube sitting on ice.
| Components | Volume | Final Concentration |
| Template DNA | 100 ng | 2 ng/µl |
| Forward primer (10 µM) | 1 - 2.5 µl | 200 - 500 nM |
| Reverse primer (10 µM) | 1 - 2.5 µl | 200 - 500 nM |
| 10X PCR buffer, with Mg2+ | 5 µl | 1X |
| 25 mM MgSO4 (optional)* | 0 - 3 µl | 1.5 - 3 mM |
| dNTP Mix (10 mM) | 1 µl | 200 µM |
| Hotstart taq DNA Polymerase (5 U/µl) | 0.5 - 1 µl | 2.5 - 5 U |
| Nuclease-free H2O | up to 50 µl | - |
* Optimal Mg2+ concentration is specific to each DNA template-primer set and
can only be determined experimentally.
• We recommend preparing a mastermix for multiple reactions to minimize
reagent loss and enable accurate pipetting.
2. Mix contents of tube and centrifuge briefly.
3. Incubate tube in a thermal cycler at 94°C for 10 mins to completely activate the
HotStart Taq Polymerase and denature the template.
4. Perform 30 - 35 cycles of PCR amplification as follows:
Denature: 94°C for 30 sec
Anneal: 45 - 72°C for 30 sec
Extend: 72°C for 1 min/1 kb template
5. Incubate for an additional 5 mins at 72°C and maintain the reaction at 4°C. The
samples can be stored at -20°C until use.
6. Analyze the amplification products by agarose gel electrophoresis and visualize
by ethidium bromide or SafeView™ (Cat No. G108) staining. Use appropriate
molecular weight standards.
