NZYSpeedy Proof DNA polymerase

NZYSpeedy Proof DNA polymerase
Catalogue numbers:
MB10901, 125 U
MB10902, 500 U
Description: NZYSpeedy Proof DNA polymerase is a recombinant thermostable DNA polymerase purified
from Escherichia coli that combines high fidelity and fast polymerization reaction. NZYSpeedy Proof DNA
polymerase possesses 3´→5´ exonuclease proofreading activity that enables the polymerase to correct
nucleotide misincorporation errors. 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 seconds is required for the successful synthesis ok 1 kb size DNA. NZYSpeedy Proof DNA
polymerase generates blunt-ended polymerase chain reaction (PCR) products that are suitable for cloning
with NZYTech´s NZYPCR cloning kit (MB01301 or MB01302).
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.
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, 1.5 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: NZYSpeedy Proof DNA polymerase has been determined to be >95% pure as judged by SDS
polyacrylamide gel electrophoresis followed by Coomassie Blue staining.
Enzyme concentration: 2.5 U/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
Functional assay: NZYSpeedy Proof DNA polymerase is tested for performance in the polymerase chain
reaction (PCR) using 1 unit of enzyme to amplify a 1000 bp region of the Escherichia coli genome. The
resulting PCR products are visualized as a single band in an agarose gel.
Nuclease assays: 20 U of NZYSpeedy Proof DNA polymerase were incubated with 0.5 μg of pUC18 for 2
hours at 37 °C or 72 °C. No nicking activity was observed after agarose gel electrophoresis.
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% (v/v) Triton X-100. 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
(1.5-2.25 mM). Vortex the MgCl2 solution thoroughly after thawing and prior to use.
Reaction Conditions
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):
Reaction buffer, 10× (provided)
MgCl2, 50 mM (provided)
1.5 µl 1
dNTP mix, 25 mM solution
0.5-0.8 μl
Primers
0.2-1 μM
Template DNA
10-100 ng
NZYSpeedy Proof DNA Polymerase (2.5 U/μl)
Nuclease-free water
1
5 μl
0.5-1 μl
up to 50 μl
For targets greater than 2 kb, final concentration of MgCl2 may be adjusted to between 1.5 and 2.2 mM.
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.
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
3. Perform PCR using the following cycling conditions (Fast PCR):
1
Step
Temperature
Time
Number of cycles
Pre-denaturation
95 °C
2 min
1
Denaturation
95 °C
20 sec
Annealing
Lowest primer Tm °C
10 sec
Extension
70 °C
15 sec/ kb
Hold
4-20 °C
20-40 1
A higher number of cycles may be necessary for genomic DNA templates
4. Separate the PCR products by agarose gel electrophoresis (0.7-1.2%, w/v) and visualize with
GreenSafe (MB08801).
Data
M 100 50 25 12.5 6.25 NC
100 50 25 12.5 6.25 NC (ng)
1 kb
2.5 U NZYSpeedy Proof
DNA polymerase
1.25 U NZYSpeedy Proof
DNA polymerase
Figure 1. Amplification of a 1 kb DNA fragment using a 15 sec extension period. Different amounts of E. coli genomic DNA were
amplified with 2.5 U or 1.25 U of NZYSpeedy Proof DNA polymerase. NC: No template control. Lane M: NZYDNA Ladder III
(MB0440).
M
1 2 3
Figure 2. Amplification of different DNA fragments using a limited extension period of 15
sec/kb. Three different DNA fragments of various sizes were amplified using a limited
extension period using 2.5 U of NZYSpeedy Proof DNA polymerase. Lane M: NZYDNA Ladder III
(MB0440); 1: 350 pb PCR product (sheep genomic DNA, 120 ng); 2: 1 kb PCR product (E. coli
genomic DNA, 100 ng); 3: 2 kb PCR product (plasmid DNA, 10 ng).
Revised: 12/10
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