(v/v) Triton X-100. Vortex the Reaction Buffer solution
thoroughly after thawing and prior to use. Repeated freezethaw cycles will affect the stability of the buffer (the buffer
will remain stable at 4 °C up to one month).
NZYSpeedy Proof DNA
polymerase
Magnesium Chloride solution: 50 mM MgCl2. Allows
users to optimize MgCl2 concentrations (1.5-2.25 mM).
Vortex the MgCl2 solution thoroughly after thawing and prior
to use.
Standard Protocol
Catalogue number:
MB10901, 125 U
MB10902, 500 U
Description
NZYSpeedy Proof DNA polymerase is a recombinant
thermostable DNA polymerase purified from Escherichia coli
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.
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):
that combines high fidelity and fast polymerization
capacities. NZYSpeedy Proof DNA polymerase possesses
Reaction buffer, 10× (provided)
5 µL
3´→5´ exonuclease proofreading activity that enables the
polymerase amplifying DNA with increased accuracy and
MgCl2, 50 mM (provided)
1.5-2.5 mM
dNTPs mix
0.25-0.5 mM
yield. The error rate of the enzyme is similar to that of Pfu
and Kod DNA polymerases and significantly lower than the
error rate of Taq DNA polymerases. NZYSpeedy Proof DNA
polymerase is highly efficient in the amplification of DNA
fragments up to 3 kb in a short reaction period. Only 15
Primers
0.2-0.5 µM
Template DNA
0.01-0.5 µg
seconds is required for the successful synthesis ok 1 kb size
DNA. NZYSpeedy Proof DNA polymerase generates bluntended polymerase chain reaction (PCR) products that are
NZYSpeedyProof DNA
Polymerase (2.5 U/µL)
0.2-0.5 µL
suitable for cloning with NZYTech´s NZY-blunt PCR cloning
kit (MB121).
Nuclease-free water
up to 50 µL
Storage temperature
NZYSpeedy Proof DNA polymerase should be stored at -20
°C in a constant temperature freezer. NZYSpeedy Proof DNA
polymerase will remain stable up to 3 years if stored as
specified.
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 the thermal cycling. Centrifuge the
reactions in a microcentrifuge for 5 seconds.
3. Perform PCR using the following parameters:
Storage buffer
20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM
DTT, 50% (v/v) glycerol.
Unit definition
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.
Enzyme concentration: 2.5 U/µL
Reaction buffer (10×): 1.2 M Tris-HCl, pH 8.0, 60 mM
(NH4)2SO4, 100 mM KCl, 0.01% (w/v) nuclease-free BSA, 1%
Cycle step
Temp.
Time
Cycles
Initial
denaturation
95 °C
120 s
1
Denaturation
95 °C
30-60 s
Annealing
*
30-60 s
Extension
70 °C
15 s/kb
Final Extension
70 °C
5-10 min
20-40
1
*Annealing temperature should be optimized for each primer
set based on the primer Tm; typically it should be Tm-5 °C.
4. Separate the PCR products by 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 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
delete 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.
Functional assay
NZYSpeedy Proof DNA polymerase polymerase is tested for
performance in a polymerase chain reaction (PCR) using 1
unit 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 the reaction buffer and MgCl2 solution.
Troubleshooting
No product amplification or low yield
• Inadequate annealing temperature
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
Quality control assays
Purity
NZYSpeedy Proof DNA polymerase purity is >90% as judged
by SDS-PAGE followed by Coomassie Blue staining.
Genomic DNA contamination
NZYSpeedy Proof DNA polymerase must be free of any
detectable genomic DNA contamination as evaluated
through PCR.
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.
Nuclease assays
0.2-0.3 µg of pNZY28 plasmid DNA are incubated with 5 U of
NZYSpeedy Proof 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 NZYSpeedy Proof DNA polymerase
buffer and MgCl2 solution.
Revised 12/12
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, Portugal
Tel.:+351.213643514
Fax: +351.217151168
www.nzytech.com