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HawkZ05 Fast DNA Polymerase
Easy-to-use polymerase for qPCR and qRT-PCR
Cat. No. 07 731 264 103
Cat. No. 07 731 329 103
1.
Solution, 40 U/␮l
Glycerol-free solution, 200 U/␮l
What this Product Does
DNA and RNA Amplification
The HawkZ05 Fast DNA Polymerase is designed for fast, highly sensitive and specific real-time PCR analysis of DNA and RNA. HawkZ05
Fast DNA Polymerase is a blend of Z05 DNA Polymerase and a specific oligonucleotide (aptamer), together providing the hot start features.
Z05 Fast DNA Polymerase is the mutated, recombinant version of the
thermostable enzyme isolated from the thermophilic eubacterium
Thermus species Z05, expressed in E. coli. In many aspects, the
enzyme is very similar to Tth DNA Polymerase, however, it exhibits a
higher stability under PCR conditions and an improved reverse transcriptase activity.
HawkZ05 Fast DNA Polymerase is suitable for detection formats such
as hydrolysis probes, hybridization probes, and SYBR Green.
The concentrated, glycerol-free formulation is ready for lyophilization
and suitable for the preparation of dry amplification mix preparations.
Content
Label
Catalog Number
HawkZ05 Fast Polymerase, 40U/␮l1)
07 731 264 103
custom fill
HawkZ05 Fast Polymerase, Gycerol-free,
200 U/␮2)
07 731 329 103
custom fill
1)
Storage buffer: 20 mM Tris/HCl, 100 mM KCl, 0.1 mM EDTA, 0.5% (v/v), 1 mM DTT,
50% (v/v) Tween 20, and glycerol, pH 8.0 at +4°C.
2)
Storage buffer: same composition as 1) without glycerol.
Storage and Stability
Store the vial at ⫺15 to ⫺25°C.
The solution is stable until the expiration date printed on the label. The
solution is also stable at +2 to +8°C for 4 weeks.
 Avoid repeated freeze/thaw cycles ( > 5 times).
How to Use this Product
2.1
Before You Begin
General Considerations
The optimal reaction conditions (concentration of template DNA or
RNA, concentration of PCR primers, incubation temperatures and
times, cycle number) depend on the specific template/primer system
and must be determined individually.
Sample Material
Use any DNA suitable for qPCR or template RNA (total RNA or mRNA)
suitable for RT-PCR, in terms of purity, concentration, and absence of
inhibitors.
1115.07819838001 Œ
Content version: November 2015
Store at ⫺15 to ⫺25°C
Buffer for PCR and RT-PCR
250 mM Tricine, 400-500 mM potassium acetate, 10-25% glycerol,
0.05% Tween 20, pH 8.0.
Application
2.
y Version 01
Enzyme Concentration
Use 5-10 U for a 20 ␮l reaction volume.
Primer Concentration
Use PCR primers at a final concentration of 0.1-1.0 ␮M.
 Always use equimolar primer concentrations.
Nucleotides
Use a concentration of 200 ␮M of each dATP, dGTP, dCTP, dTTP (or
300 ␮M of dUTP instead of dTTP when including UNG).
Probes
As a starting point, use a probe concentration of 0.25 ␮M each.
However, suitable concentrations range from 0.1 to 0.2 ␮M.
 The optimal probe concentration is the lowest concentration that
results in the lowest quantification cycle value (Cq) and adequate
fluorescence dynamics for a given target concentration.
 To ensure efficient probe cleavage, the Tm of the hydrolysis probe
should be higher than the Tm of the primers.
Salt
Use magnesium or manganese acetate for PCR reactions.
 For RT-PCR reactions, use only manganese acetate.
A recommended starting concentration is 1.5 mM. The range varies
from 1.0 mM to 4.0 mM. Low manganese or magnesium ion concentrations may result in poor yield and too much may produce nonspecific products.
Prevention of Carryover Contamination
Uracil-DNA Glycosylase* (UNG) can be used for preventing carryover
contamination during PCR and RT-PCR. This technique involves incorporating dUTP into amplification products, permitting pretreatment of
subsequent PCR mixtures with UNG.
Add 1-2 U to a 20 ␮l RT-PCR mixture.
If using UNG, perform the following pre-incubations:
- 50°C, 2-5 min to degrade contaminating amplicon.
- 95°C, 5 s to inactivate UNG.
DMSO
1-5% can be used for further optimization.
Negative Control
Always run a negative control with the samples. To prepare a negative
control, replace the template DNA or RNA with water, PCR Grade.
custombiotech.roche.com
2.2
Standard Procedure for qRT-PCR
Following the operator's manual of the instrument supplier, program
the PCR profile with the parameters indicated below:
Product
Cat. No.
Cycles
Analysis
Mode
Target
Temp.
Hold
Time
Program
Name
Uracil-DNA Glycosylase
11 780 565 103
1
None
+60°C to
+65°C 3)
5 to
10 min 4)
Reverse
transcription
Changes to Previous Version
First Version
1
None
+95°C
5s
Initial
denaturation
45
Quantification
+95°C
5s
Amplification
(2 steps)
Trademarks
HAWKZ05 is a trademark of Roche. All other product names and
trademarks are the property of their respective owner.
None
+4°C
1
+60°C
3)
30 s
Cooling
3)
Z05 enzyme activity is inhibited by the aptamer at temperatures below 55°C. At temperatures above 60°C, the aptamer fully releases the polymerase and full enzyme activity is
achieved. Make sure that the reverse transcriptase and PCR primers have sufficient stability to bind template at the reverse transcription step (hold temperature) and the annealing
step.
4)
Ten minutes is the recommended default protocol. Depending on your assay, it should be
possible to reduce the time for reverse transcription down to 4 minutes. For best results,
run a test with 10, 8, 6, 4 minutes, etc.
3.
Additional Information on this Product
Ordering Information
Regulatory Disclaimer
For further processing only.
License Limitations
NOTICE: For patent license limitations for individual products, please
refer to www.technical-support.roche.com.
Online Technical Support
Please visit our Online Technical Support site for additional information about this product: www.technical-support.roche.com
Hot Start Mechanism
The aptamer/polymerase mixture is a hot start system with reversible
inhibition of the polymerase activity at lower temperatures. Polymerase
inactivation is achieved by a tight bond of the folded aptamer-oligonucleotide to the active site of the polymerase at lower temperatures.
Upon heating above +60°C, the aptamer acts like a molecular switch,
changing its temperature-dependent tertiary structure and releasing
the active polymerase. Dropping the temperature below +55°C shuts
off the polymerase activity again. Similar to antibody-based methods,
the enzyme is much more quickly activated by heating, than chemically modified polymerases. In contrast to antibodies, the aptamer-oligonucleotide is much more stable, allowing longer storage at room
temperature.
Quality Control
Each lot of HawkZ05 Fast Polymerase is tested to meet specifications
for enzyme activity, aptamer concentration, and contaminating activities.
4.
Supplementary Information
Text Conventions
To make information consistent and understandable, the following text
conventions are used in this document:
Text Conventions
Use
Numbered Instruc- Steps in a procedure that must be performed
tions labeled 쐃, 쐇, in the order listed.
etc.
Asterisk *
Denotes a product available from Roche
Diagnostics.
Symbols
In this document, the following symbols are used to highlight important information:
Symbol Description

Information Note:
Additional information about the current topic or procedure.

Important Note:
Information critical to the success of the procedure or use
of the product.
For more information about this product, as well as documentation such as Instructions for Use and Safety Data Sheets, please
visit custombiotech.roche.com
For more information, contact your local Roche representative.
Europe, Middle East,
Africa, and Latin America:
Roche Diagnostics
Deutschland GmbH
Phone +49 621 759 8580
Fax
+49 621 759 6385
mannheim.custombiotech@
roche.com
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Roche Diagnostics K.K
Phone +81 3 5443 5285
Fax
+81 3 5443 7934
japan.custombiotech@roche.
com
Asia Pacific:
Roche Diagnostics Asia
Pacific Pte. Ltd
Phone +65 6371 6638
Fax
+65 6371 6601
apac.custombiotech@roche.
com
Canada:
Roche Diagnostics
Phone +1 450 686 7050
Fax
+1 450 686 7012
custombiotech.can@roche.
com
Roche Diagnostics GmbH
Sandhofer Strasse 116
68305 Mannheim
Germany
United States:
Roche Diagnostics
Corporation
Phone +1 800 428 5433,
ext. 14649 (toll-free)
Fax
+1 317 521 4065
custombiotech.ussales@
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