Supreme NZYTaq 2× Green Master Mix

Supreme NZYTaq 2× Green Master Mix
Catalogue numbers:
MB05401, 100 U
MB05402, 500 U
Description: Supreme NZYTaq 2× Green Master Mix is a premixed ready to use solution containing 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.
In addition, the master mix contains dNTPs, MgCl2 and reaction buffer at optimal concentrations for
efficient amplification of DNA templates by PCR. MgCl2 final concentration is 2.5 mM, allowing the
implementation of a comprehensive range of PCR protocols. Reactions assembled with Supreme NZYTaq
2× Green Master Mix maybe directly loaded onto agarose gels. Supreme NZYTaq 2× Green Master Mix
contains two dyes (blue and yellow) that allow monitoring the progress of electrophoresis. Supreme
NZYTaq 2× Green Master Mix is not suitable when direct fluorescent or absorbance readings are required
without prior purification of the amplified DNA from PCR.
Supreme NZYTaq DNA 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 to 5-10 pg. The activated
enzyme maintains the same functionality as Taq DNA polymerase. Supreme NZYTaq DNA polymerase
lacks 3´→5´ exonuclease activity. Resulting polymerase chain reaction (PCR) products have an A overhang
and are suitable for cloning with NZYTech´s NZYPCR cloning kit (MB01301 or MB01302).
Storage temperature: Supreme NZYTaq 2× Green Master Mix should be stored at -20 °C, in a constant
temperature freezer. Minimize the number of freeze-thaw cycles by storing in working aliquots. The Mix
maybe stored at 4 °C for up to 7 weeks.
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.
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
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: 0.2 U/µl
Functional assay: Supreme NZYTaq 2× Green Master Mix 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.
Supreme NZYTaq Green Master Mix (2×
×): NZYTech’s Master Mix has a proprietary formulation.
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):
Supreme NZYTaq Green Master Mix, 2×
Primers
0.05-0.2 µM1
Template DNA2
0.0005-0.5 µg
Nuclease-free water
1
2
25 µl
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.
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
Example Amplifications
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 standard 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.
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