Taq DNA Polymerase
- separate MgCl2 Solution Concentration
Storage Temperature
5 U/µl
–20 °C
Ordering Information
Taq DNA Polymerase
Order No. 3020302300
Order No. 3020302200
Order No. 3020302400
100 Units
1000 Units
2500 Units
Description
Recombinant Taq DNA polymerase (Thermus aquaticus YT1) purified from Escherichia coli DH1 supplied with 10x
NH4 Reaction Buffer, 10x KCL Reaction Buffer and separate magnesium solution.
Components
Enzyme storage buffer
20 mM Tris-HCl pH 8.0 (at 25°C), 100 mM KCl, 0.1 mM EDTA, 1mM DTT, 50 % glycerol and 0.5% Tween 20.
10x NH4 Reaction Buffer
500 mM Tris-HCl pH 8.8 (at 25°C), 160 mM (NH4)2SO4, 0.1% Tween 20
10x KCL Reaction Buffer
500mM KCl, 100 mM Tris-HCl pH 8.8 (at 25°C), 15mM MgCl2, 0.1% Tween 20
MgCl2 Solution
50 mM MgCl2, recommended range for final concentration: 1.0 mM - 3.0 mM
Unit definition
One unit is defined as the amount that incorporates 10 nmoles of dNTP´s into acid insoluble form in 30 minutes at
74°C under the assay conditions, using reaction conditions: 25 mM TAPS (N-tris-(hydroxymethyl)-methyl-3-aminopropanesulfonic acid, sodium salt) pH 9.3 (at 25°C); 50 mM KCl; 2 mM MgCl2; 1 mM ß-mercaptoethanol; 200 µM
32
each dATP, dGTP, dTTP; 100 µM dCTP (a mix of unlabelled and [α- P]-labelled); 12.5 µg activated salmon sperm
DNA in a final volume of 50 µl.
Storage/Stability
Store Taq DNA Polymerase at -20°C in a constant temperature freezer. Taq DNA Polymerase will remain stable
at least for one year.
Precautions and Disclaimer
Taq DNA Polymerase is for research use only. Not recommended or intended for diagnosis of disease in humans
or animals. Do not use internally or externally in humans or animals.
Quality Control
Endonuclease and exonuclease activities were not detectable after 2 and 1 hours incubation, respectively, of 1 µg
lambda DNA and 0.22 µg of EcoRI-digested lambda DNA at 72°C in the presence of 15-20 units of Taq DNA
Polymerase.
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Taq DNA Polymerase 04/2013
Important Parameters
Template
The template should be of high molecular weight and its purity and integrity checked on an agarose gel prior to use.
2+
Contamination of the DNA template with RNA can result in reduced yield in PCR due to chelating of Mg and hence
should be pre-purified. Template concentration will have a profound effect on PCR performance. Recommended
amounts for standard PCR and reaction volume 25 µl are:
•
•
•
Human DNA up to 500 ng
Plasmid DNA 0.1–10 ng
Bacterial DNA 1–10 ng
Difficult Templates
For better amplification results use additional substances for example glycerol (5 – 10 %), DMSO (2 – 20 %),
formamide (2 – 20 %), tetramethylammonium chloride (0.01 – 10 mM) betaine (up to 1M) or combinations of these.
The role of DMSO, betaine and glycerol can significantly reduce the presence of non-specific binding products, which
results in band smearing and presence of non-target sequence products.
Basic Protocol
Mix the following components on ice in a thin-walled 0.2 ml reaction tube:
Component
Double destilled, sterile water
Volume
Final concentration_
variable (added to the final
volume of 50 µl)
10x NH4 Reaction Buffer or
10x KCl Reaction Buffer
5 µl
50 mM MgCl2 Solution
1x
1.5 µl
50x dNTP-Mix (each 12.5 mM)
1.5 mM
1 µl
0.25 mM
Primer A
variable
0.2 – 1 µM
Primer B
variable
0.2 – 1 µM
Taq DNA Polymerase (5 U/µl)
Template DNA
0.4 – 0.8 µl
2 – 4 Units/ 50 µl __
variable
≤1 µg____________
Total volume
50 µl
- Mix the MgCl2 Solution before use by vortexing vigorously.
Reaction conditions (incubation temperatures and times, concentrations of template DNA, primers, magnesium ions
2+
and enzyme) depend on template and primers used. Optimal Mg concentrations vary between 1-4 mM and have to
2+
be determined empirically. However, many applications work at the standard concentration of 1.5 mM Mg .
2+
Advanced applications on genomic DNA require higher Mg concentrations (2-3 mM) adjustable with the separate
MgCl2 solution supplied with the set.
Cycling conditions
Temperature for chain elongation:
2+
Best Mg concentration for specific activity:
2+
Mg concentration range for PCR:
Half life in PCR:
Extension rate:
Longest product:
Processivity:
5’-3’ exonuclease activity:
3’-5’ exonuclease activity (proofreading):
Error rate/fidelity:
Amount used per reaction:
Routine application range:
Favoured application:
70-72°C
10 mM
1-3 mM
25 cycles
1-2 kb
10 kb
40-50 bases
Yes
No
-5
2.7 x 10
0.2-2.5 U
amplification of DNA fragments ranging from
0.1 kb to 5 kb
standard PCR applications which do not require
a high synthesis fidelity
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Troubleshooting
Observation
Check
nonspecific products
- concentration of enzyme, and/or primer was too high
(smearing)
- annealing temperature for primers
- too many cycles
- annealing and extension time too long
- too much template DNA
low yield of product
- not enough or to much enzyme
- concentration of dNTPs was too high
- denaturation/extension temperature too high
- incorrect annealing temperature
- too few cycles
- poorly designed primers
- inhibitors from DNA purification (i.e. SDS)
no product
- incorrect annealing temperature
- incomplete denaturation
- poorly designed primers
- use of destroyed components due to wrong storage
Technical Assistance
If you have any questions or problems regarding any aspects of Taq DNA Polymerase or other STRATEC Molecular
products, please do not hesitate to contact us. For technical support or further information in Germany please dial
+49 (0)30 9489-2901 or +49 (0)30 9489-2910. For other countries please contact your local distributor.
STRATEC Molecular GmbH
Robert-Rössle-Str. 10
13125 Berlin
Phone: +49 30 9489 2901/2910
Fax: +49 30 9489 2909
E-mail: info.berlin@stratec.com
www.stratec.com
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Taq DNA Polymerase 04/2013