Supreme NZYTaq DNA polymerase

Supreme NZYTaq DNA polymerase
Catalogue numbers:
MB07901, 500 U
MB07902, 1000 U
MB07903, 2500 U
Description: Supreme NZYTaq DNA polymerase contains a recombinant modified form of Taq DNA
polymerase with a hot-start like PCR capacity. The enzyme is inactive at room temperature, avoiding
extension of non-specifically annealed primers or primer-dimers and providing higher specificity of DNA
amplification. The functional activity of the enzyme is restored during a short 5-minute incubation step at
95 °C. This highly robust Taq polymerase is a broad range enzyme suitable for a variety of general PCR
applications to amplify target DNA sequences up to 3 kb in size. It enhances many complex PCR reactions
by increasing both specificity and yield in a range of DNA concentrations up 5-10 pg (Figures 1 and 2). The
activated enzyme maintains the same functionality as Taq DNA polymerase. Supreme NZYTaq DNA
polymerase lacks 3´→5´ exonuclease activity. Thus, resulting polymerase chain reaction (PCR) products
have an A overhang and are suitable for cloning with NZYTech´s NZY-A PCR cloning kits (MB05301 or
MB05302). Supreme NZYTaq DNA polymerase allows an easy handling and a room-temperature setup.
Figure 1. Increased yield in DNA amplification with Supreme
NZYTaq DNA polymerase. Amplification of a 1.3 kb DNA
fragment using a 2-fold E. coli genomic DNA dilution ranging
from 240 to 15 pg (lanes 1-5) with NZYTaq DNA polymerase or
with Supreme NZYTaq DNA polymerase. Lane M (Marker):
NZYDNA Ladder III. Results demonstrate an increase in the yield
of the desired product in a range of DNA concentrations up to 15
pg when Supreme NZYTaq DNA polymerase is used.
Figure 2. Increased specificity and yield in DNA amplification
with Supreme NZYTaq DNA polymerase. Amplification of a 350
bp DNA fragment using a 2-fold sheep genomic DNA dilution
ranging from 200 to 25 pg (lanes 1-4) with standard NZYTaq DNA
polymerase or with Supreme NZYTaq DNA polymerase. Lanes 5
contain no DNA (controls). Lane M (Marker): NZYDNA Ladder V.
Results demonstrate a shift from mainly primer-dimers to the
desired product in a range of DNA concentrations up to 25 pg
when Supreme NZYTaq DNA polymerase is used.
NZYTech, Lda. Estrada do Paço do Lumiar, Campus do Lumiar, Edifício E – R/C, 1649-038 Lisboa, Portugal
Tel: +351 213643514 Fax:+351 217151168 [email protected] www.nzytech.com
Storage temperature: Supreme NZYTaq DNA polymerase should be stored at -20 °C, in a constant
temperature freezer. Supreme 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.
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 of
unlabelled and α-[32P]-labelled), 10 μg activated salmon sperm DNA in a final volume of 50 μl.
Purity: Supreme NZYTaq DNA polymerase has been determined to be >95% pure as judged by SDS
polyacrylamide gel electrophoresis followed by Coomassie Blue staining.
Enzyme concentration: 5 U/μl
Functional assay: Supreme NZYTaq DNA polymerase is tested for performance in the polymerase chain
reaction (PCR) using 1 unit of enzyme to amplify a 924 bp region of the glutaminase gene from 100
molecules of Escherichia coli genomic DNA. The resulting PCR product is visualized as a single band in an
ethidium bromide-stained agarose gel.
Nuclease assays: 20 U of Supreme NZYTaq DNA polymerase were incubated with 0.5 μg of pUC18 for 2
hours at 37 °C and 72 °C. No nicking activity was observed after agarose gel electrophoresis.
Reaction buffer (10×
×): 670 mM Tris-HCl, pH 8.8, 160 mM (NH4)2SO4, 400 mM KCl, 0.1% 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: 50 mM MgCl2. Provided to allow users to optimize MgCl2 concentrations
(1.5-4.0 mM). Vortex the MgCl2 solution thoroughly after thawing and prior to use.
NZYTech, Lda. Estrada do Paço do Lumiar, Campus do Lumiar, Edifício E – R/C, 1649-038 Lisboa, Portugal
Tel: +351 213643514 Fax:+351 217151168 [email protected] www.nzytech.com
Reaction Conditions
1. At room temperature, 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):
Reaction buffer, 10× (provided)
5 μl
MgCl2, 50 mM (provided)
1.5-4.0 mM
dNTP mix, 25 mM solution
0.5-1.0 μl
Primers
Supreme NZYTaq (5 U/μl)
Template DNA2
Nuclease-free water
1
2
0.05 - 0.2 μM1
0.5-1.0 μl
0.0005-0.5 μg
up to 50 μl
The efficiency of Supreme NZYTaq may be compromised at higher primer concentrations.
Template DNA should be the last component to be added to the reaction mixture.
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 standard parameters with an initial 5-minutes denaturation step at 95 °C. The
extension time should be at least 1 minute/kb target length (72 °C). Annealing temperatures may
need to be optimized for each primer set based on the primers Tm.
4. Separate the PCR products by agarose gel electrophoresis (0.7-1.2%, w/v) and visualize with ethidium
bromide or any other means.
Revised: 11/10
NZYTech, Lda. Estrada do Paço do Lumiar, Campus do Lumiar, Edifício E – R/C, 1649-038 Lisboa, Portugal
Tel: +351 213643514 Fax:+351 217151168 [email protected] www.nzytech.com