Standard Protocol
NZYTaq DNA polymerase
The following standard protocol serves as a general guideline
and a starting point for any PCR amplification. Optimal
reaction conditions (incubation times and temperatures,
concentration of DNA polymerase, primers, MgCl2, and
template DNA) vary and may need to be optimized.
Catalogue number:
1. On ice, in a sterile, nuclease-free microcentrifuge tube,
prepare a reaction mixture for the appropriate number of
samples to be amplified. A single reaction mixture should
combine the following components (for a 50 μL reaction):
MB00101, 500 U
MB00102, 1000 U
MB00103, 2500 U
Description
NZYTaq DNA polymerase is an ultrapure recombinant
modified form of Taq DNA polymerase purified from
Escherichia coli and supplied with an optimized 10× Reaction
Buffer and a 50 mM MgCl2 solution. NZYTaq DNA
polymerase lacks 3´5´ exonuclease activity and supports
robust and reliable reactions while tolerating a wide range of
templates. Resulting polymerase chain reaction (PCR)
products have an A overhang and are suitable for cloning
with NZYTech´s TA PCR cloning kits (MB053 or MB137).
Storage temperature
NZYTaq DNA polymerase should be stored at -20 °C in a
constant temperature freezer. NZYTaq DNA polymerase will
remain stable up to 3 years if stored as specified.
Storage buffer
20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM
DTT, 50% (v/v) glycerol.
Reaction buffer, 10× (provided)
5 μL
MgCl2, 50 mM (provided)
1.5-4.0 mM
dNTPs mix
0.25-0.5 mM
Primers
0.2-0.5 μM
Template DNA
0.01-0.5 μg
NZYTaq (5 U/μL)
0.2-0.5 μL
Nuclease-free water
up to 50 μL
2. If using a thermal cycler without a heated lid, overlay the
reaction mix with 1-2 drops of mineral oil to prevent
evaporation during thermal cycling. Centrifuge the reactions
in a microcentrifuge for 5 seconds.
3. Perform PCR using standard parameters.
Cycle step
Temp.
Time
Cycles
Initial
denaturation
95 ºC
120 s
1
Denaturation
95 ºC
30-60 s
Unit definition
Annealing
*
30-60 s
One unit is defined as the amount of enzyme required to
catalyse the incorporation of 10 nmoles of dNTPs into acid
insoluble material in 30 minutes at 72 °C, under the following
assay conditions: 50 mM Tris-HCl, pH 9.0, 50 mM NaCl, 2.5
mM MgCl2, 200 μM each of dATP, dCTP, dGTP, dTTP (a mix
32
of unlabelled and α- P-labelled), 10 μg activated salmon
sperm DNA in a final volume of 50 μL.
Extension
72 ºC
30-60 s/kb
Final Extension
72 ºC
5-10 min
Enzyme concentration: 5 U/μL
Reaction buffer (10x): 670 mM Tris-HCl, pH 8.8, 160 mM
(NH4)2SO4, 0.1% (v/v) Tween 20. Vortex the reaction buffer
solution thoroughly after thawing and prior to use. Repeated
freeze-thaw cycles will affect the stability of the buffer (the
buffer will remain stable at 4 °C up to one month).
Magnesium Chloride solution: the provided 50 mM
MgCl2 solution allows users to optimize MgCl2
concentrations. Vortex the MgCl2 solution thoroughly after
thawing and prior to use.
25-35
1
*Annealing temperature should be optimized for each primer
set based on the primer Tm; typically it should be Tm-5 ºC.
4. Separate the PCR products through agarose gel
electrophoresis (0.7-1.2%, w/v) and visualize with GreenSafe
(MB088) or any other means.
Primer Design
PCR primers generally range in length from 15–30 bases and
are designed to flank the region of interest. Primers should
contain 40–60% GC, and care should be taken to avoid
sequences that might produce internal secondary structure.
The 3´-ends of the primers should not be complementary to
avoid the production of primer-dimers. Primer-dimers
unnecessarily remove primers from the reaction and result in
an unwanted polymerase reaction that competes with the
desired reaction. Avoid three G or C nucleotides in a row near
the 3´-end of the primer, as this may result in non-specific
primer annealing, increasing the synthesis of undesirable
reaction products. Ideally, both primers should have nearly
identical melting temperatures (Tm); in this manner, the two
primers should anneal at roughly the same temperature.
Troubleshooting
No product amplification or low yield
 Inadequate annealing temperature
DNA template
The required concentration of DNA template depends on the
type of DNA being amplified. Generally 50–500 ng of
genomic DNA template is recommended. Lower amounts of
DNA template (typically 1–50 ng) can be used for
amplification of lambda or plasmid DNA or even 10-100 ng
for amplification of multicopy chromosomal genes.
Quality control assays
Purity
NZYTaq DNA polymerase purity is >90% as judged by SDS
polyacrylamide gel electrophoresis followed by Coomassie
Blue staining.
Genomic DNA contamination
NZYTaq DNA polymerase must be free of any detectable
genomic DNA contamination as evaluated through PCR.
Nuclease assays
0.2-0.3 μg of pNZY28 plasmid DNA are incubated with 5 U of
NZYTaq DNA polymerase, in 1× Reaction Buffer, for 14-16
hours at 37 °C. Following incubation, the DNA is visualised on
a GreenSafe-stained agarose gel. There must be no visible
nicking or cutting of the nucleic acid. Similar tests are
performed with NZYTaq buffer and MgCl2 solution.
The reaction mix composition may affect the melting
properties of primers and DNA. Adjust the annealing
temperature to accommodate the primer with the lowest
melting temperature (5 ° to 10 °C lower than Tm).
 Presence of PCR inhibitors
Some DNA isolation procedures, particularly genomic
DNA isolation, can result in the co-purification of PCR
inhibitors. Reduce the volume of template DNA in
reaction or dilute template DNA prior to adding to the
reaction. Diluting samples even 1:10,000 has been shown
to be effective in improving results, depending on initial
DNA concentration.
 Additives required
Adding PCR-enhancing agents (NZYTaq 5× Optimizer
Solution – MB060 or NZYTaq 2× GC-Enhancer Solution –
MB143) may improve yield while amplifying difficult
templates.
Revised 12/12
Functional assay
NZYTaq DNA polymerase is tested for performance in a
polymerase chain reaction (PCR) using 5 U of enzyme for the
amplification of a 1000 bp fragment of the glucokinase gene
from Escherichia coli genomic DNA. The resulting PCR
product is visualized as a single band in a GreenSafe stained
agarose gel. Similar functional tests are performed with
NZYTaq buffer and MgCl2 solution.
Certificate of Analysis
Test
Result
Enzyme purity*
Pass
Genomic DNA contamination*
Pass
DNase contamination
Pass
Functional assay
Pass
*These assays were exclusively performed with the enzyme
Approved by:
José Prates
Senior Manager, Quality Systems
Estrada do Paço do Lumiar, Campus do Lumiar, Edifício E, R/C, 1649-038 Lisboa
Tel: +351.213643514 Fax: +351.217151168 www.nzytech.com