ABMG078
Precision Taq DNA Polymerase PCR, 500 U (100 ul) Protocol

Precision DNA Polymerase PCR, 500 U (100 ul)

• must be stored at -20° C

Precision™ Taq DNA Polymerase is a thermostable enzyme that replicates DNA at
70°C to 80°C. It catalyzes the polymerization of nucleotides into duplex DNA in the 5’
to 3’ direction in the presence of magnesium. The enzyme has a molecular weight of
approximately 90 kDa. Unlike Taq DNA Polymerase, Precision™ Taq DNA Polymerase
exhibits 3’ to 5’ exonuclease (proofreading) activity, that enables the polymerase
to correct nucleotide incorporation errors. The error rate of Precision™ Taq DNA
Polymerase in PCR is 2.6x10-6 per nucleotide per cycle. The enzyme can be used in
PCR applications that demand high fidelity and can amplify templates up to 6 kb in
length. The amplified PCR products are blunt-ended.

Product Components	500 U
Precision™ Taq DNA Polymerase (5 U/µl)	100 µl
5X PCR buffer, with Mg2+	2 ml
25 mM MgSO4	1 ml
5X GC Enhancer	1 ml

Storage Buffer Components
50 mM Tris-HCl (pH 8.2 at 25° C), 0.1 mM EDTA, 1 mM DTT, 0.05 % CHAPS and 50 %glycerol.

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, Precision™ Taq DNA Polymerase should be stored at -20°C. Avoid
repeated freeze-thaw cycles of all Precision™ Taq components to retain maximum
performance. All Precision™ 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
5X PCR buffer, with Mg2+	10 µl	1X
25 mM MgSO4 (Optional)*	0 - 3 µl	1.5 - 3 mM
dNTP Mix (10 mM)	1 µl	200 µM
PrecisionTM Taq DNA Polymerase (5 U/µl)	0.5 - 1 µl	2.5 - 5 U
5X GC Enhancer (Optional)**	10 µl	1X
Nuclease-free H2O	up to 50 µl	-

* Optimal Mg2+ concentration is specific to each DNA template-primer set and
can only be determined experimentally.
** 5X GC Enhancer is optional and is only recommended for PCR amplification of
GC-rich DNA templates.
• 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 3 mins to completely denature the
template.
4. Perform 30 - 40 cycles of PCR amplification as follows: 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.