Download Directions for Use Taq DNA Polymerase, 500U

Directions for Use
Taq DNA Polymerase, 500U
Code
Description
Size
1B1634-1KU
1B1634-0.1KU
Uracil DNA Glycosylase (UNG) Cod
1KU-1000units (1 Unit/ul)
0.1KU-100 Units (1Unit/ul)
General Information
Uracil-DNA Glycosylase (UNG), Cod is a thermolabile recombinant enzyme produced in E. coli
(ung-) by a modified ung gene derived from Atlantic Cod. It degrades uracil-containing singleand double-stranded DNA, but not RNA or thymidine-containing DNA, by hydrolyzing the Nglycosidic bond between deoxyribose sugar and the base in uracil. This generates alkalinesensitive apyramidinic sites in the DNA that will be cleaved upon a combination of alkaline
conditions and high temperature.
Pretreatment of samples with UNG prevents PCR carryover contamination in labs that
substitute dUTP in place of dTTP during all amplification reactions. PCR products containing
uracil become substrates for UNG and will be degraded if they are present in subsequent
reaction mixtures subjected to UNG treatment. Only DNA templates containing thymidine are
not degraded by the treatment and will be amplified.
Recombinant cod UNG is irreversibly inactivated upon heating, which enables long-term storage
and subsequent analysis of post-PCR amplicons in applications such as cloning and
sequencing. AMRESCO’s UNG is compatible with PCR, qPCR and one-step qRT-PCR and
works in all commercially available master mixes. All amplification reactions must use dUTP
containing dNTP mixtures in order for the UNG decontamination method to be effective.
 Complete, irreversible heat-inactivation
 Prevents carryover contamination in PCR, qPCR and qRT-PCR
 Effective with only a 5 minute incubation step
Storage/Stability
Product is stable at least 2 years when stored at 0 to -20°C. Product may be stored at 4C° up to
6 months.
Product Use Limitations
For research use only. Not for therapeutic or diagnostic use.
AMRESCO, LLC Corporate Headquarters, 6681 Cochran Road Solon, OH 44139
Directions for Use
Protocol/Procedure:
Contamination Control in PCR and qPCR
Note: Ensure that dUTP is used instead of dTTP for PCR reactions.
The UNG storage buffer contains 20 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 0.1% (w/v)
Triton-X-100 and 50% (w/v) Glycerol.
1.
2.
3.
4.
Add 0.25 U of UNG directly to a 25 µL PCR Reaction.
Incubate the reaction 5 minutes at room temperature (20-25°C).
Perform the PCR amplification.
PCR product may be stored at -20°C or 4°C indefinitely until required for subsequent
analysis.
Contamination Control in One-step qRT-PCR
Note: The working temperature range for AMRESCO’s UNG is 20-40°C, with inactivation of
activity at 45°C and complete, irreversible inactivation at 55°C. Therefore, AMRESCO’s UNG is
compatible with one-step reverse transcriptase qPCR reactions, since it will not actively degrade
the uracil that is being incorporated into the cDNA during reverse transcription.
1. Add 0.25 U of UNG directly to a 25 µL qRT-PCR reaction.
2. Incubate the reaction 5 minutes at room temperature (20-25°C).
3. Reverse transcribe RNA at 50-55°C.
4. Perform the PCR amplification.
5. qRT-PCR product may be stored at -20°C or 4°C indefinitely until required for subsequent
analysis.
Frequently Asked Questions
What are the advantages of AMRESCO’s Uracil-DNA Glycosylase (UNG), Cod?
With complete, irreversible inactivation, AMRESCO’s UNG will not degrade new dUTPcontaining PCR amplicons, so that they may be used in cloning, sequencing, restriction
digestion, etc.
AMRESCO’s UNG is inactive at temperatures ≥ 45°C, which enables pre-treatment of one-step
reverse transcription reactions with the enzyme prior to the cDNA synthesis step. Unlike other
Uracil-DNA Glycosylases, AMRESCO’s recombinant cod UNG will not degrade the cDNA,
which will have incorporated dUTP. It will only degrade contaminating PCR amplicons in the
pretreatment step.
AMRESCO, LLC Corporate Headquarters, 6681 Cochran Road Solon, OH 44139
Directions for Use
The enzyme is active at room temperature and can prevent carryover contamination in
amplification reactions with a fast 5 minute pretreatment.
What is the source of AMRESCO’s UNG?
The recombinant cod UNG is produced in an ung negative E. coli strain and is designed to
replace the native UNG enzyme originating in Atlantic cod (Gadus morhua).
What is the stability of recombinant cod UNG?
UNG can tolerate multiple freeze-thaw cycles. It may also be stored up to 6 months at 4°C.
How should UNG be heat inactivated?
This enzyme is completely and irreversibly inactivated by incubation for 20 minutes at 55°C or at
95°C for 1 second.
How is a Unit of Cod UNG defined?
One Unit will release 1 nmol uracil from uracil-containing DNA per hour at 37°C.
Can AMRESCO’s UNG replace E. coli UDG in all applications?
AMRESCO’s UNG can replace E. coli UDG in all applications except those that require activity
above 40°C.
Why must dUTP be used in place of dTTP in every amplification reaction?
dUTP-containing DNA will be degraded by UNG, but dTTP- containing DNA will not. If
amplicons with dTTP are present in the lab, they may potentially contaminate future reactions
because they will not be degraded by UNG treatment.
What is the advantage of a completely, irreversibly heat-inactivated UNG?
UNG (or UDG) enzymes from other sources, which are not irreversibly inactivated, eventually
reactivate and degrade the new dUTP-containing amplicons even without the deliberate addition
of more UNG. AMRESCO’s UNG is irreversibly heat-inactivated, ensuring that the amplicon
may be stored or used in further applications without being degraded. The sample will remain
intact unless it is deliberately treated with UNG.
What are the buffer and temperature requirements for UNG activity?
UNG is active in all common PCR, qPCR and qRT-PCR buffers and master mixes. Its activity in
common Taq buffers with 1.5 – 4 mM MgCl2 is close to 100%. The optimal salt concentration is
50 mM NaCl, while excess salt decreases enzyme activity. UNG is active between pH 7-9, but
is optimal at pH 7.5. Effective temperatures range from 20-40°C, with optimal activity at 37°C.
AMRESCO, LLC Corporate Headquarters, 6681 Cochran Road Solon, OH 44139
Directions for Use
How much UNG should be added per reaction?
Typically, 0.1-1 U per 50 µL reaction works well, but each application is different and the amount
should be determined empirically.
What is the molecular weight of AMRESCO’s recombinant cod UNG?
UNG is a 28 kDa protein.
Does Uracil Glycosylase Inhibitor (UGI) inhibit UNG?
AMRESCO’s UNG does not require inhibitors due to its irreversible inactivation at 55°C.
However, it is does bind UGI in a 1:1 ratio and will be inhibited by it.
Will UNG remove uracil from both ss- and dsDNA with the same efficiency?
No, UNG removes uracil from ss-DNA at a rate of 200% compared to dsDNA.
Will UNG remove uracil from RNA? Will it work with short oligos (e.g. 25-mer)?
UNG does not act on uracil on the ribose sugar backbone. It will work on uracil-containing
oligos as small as 5-mers.
What is the difference between UNG and UDG?
The E. coli enzyme Uracil DNA Glycosylase, which may also be called Uracil-N-Glycosylase, is
abbreviated frequently as UDG or UNG. Both names and abbreviations refer to the protein
encoded by the ung gene and are correct. Although AMRESCO’s recombinant UNG is derived
from Atlantic cod, the E. coli enzyme was the first to be identified.
Does UNG eliminate contamination 100% in PCR?
UNG does degrade all uracil-containing DNA from prior PCR reactions, but may be less efficient
at decontaminating very small amplicons containing few uracils. Standard precautions for
preventing PCR contamination should still be in use, as new reactions may be contaminated at
any time by DNA lacking uracil.
How should PCR reactions treated with UNG be stored after amplification?
UNG is irreversibly heat inactivated before the completion of PCR amplification, enabling the
long-term storage of new uracil-containing PCR products without the need for product
purification or concern about degradation. The PCR product may be stored without time or
temperature limitations and may be used directly in downstream applications, such as cloning or
sequencing.
Can uracil-containing DNA be used for restriction digests and molecular cloning?
Yes, uracil-containing DNA may work in restriction digestion if the required restriction enzyme is
capable of digesting dUTP-containing DNA. For example, EcoRI and BamHI will work well with
dUTP-containing DNA, whereas HindII and HindIII show reduced activity. Cloning of dUTPcontaining DNA works as well as it does for dTTP-containing DNA in cloning, but should be
done using ung- bacterial hosts.
AMRESCO, LLC Corporate Headquarters, 6681 Cochran Road Solon, OH 44139
Directions for Use
Can uracil-containing DNA be used in analysis of protein-DNA interactions?
dUTP-containing DNA may interfere with protein binding or DNA-protein interaction studies.
Can uracil-containing DNA be used for sequencing?
dUTP-containing DNA performs as well as dTTP-containing DNA in dideoxy sequencing.
Can uracil-containing DNA be used as a probe for hybridization?
Yes, it will perform as well as DNA with dTTP in hybridization.
Does uracil-containing DNA migrate normally in electrophoresis and can it be visualized
with DNA stains?
Yes, it will migrate on gels in the same way and will be stained with the same efficiency as
dTTP-containing DNA.
How can there be a DNA band in a negative control even when the PCR reactions were all
treated with UNG?
Although UNG will degrade all uracil-containing DNA, it is still possible to have contamination
from non-substrate DNA containing thymidine. This contamination may be introduced from
pipettes, contaminated reagents, etc. Always use standard precautions for preventing
contamination.
References:
1. Purification and characterization of a cold-adapted uracil-DNA glycosylase from Atlantic cod
(Gadus morhua). Lanes O., et al. (2000) Comparative Biochemistry and Physiology – Part
B: Biochemistry & Molecular Biology. 127: 399-410.
2. Identification, cloning, and expression of uracil-DNA glycosylase from Atlantic cod (Gadus
morhua): characterization and homology modeling of the cold-active catalytic domain.
Lanes O., et al. (2002) Extremophiles. 6: 73-86.
3. Transcripts of developmentally regulated Plasmodium falciparum genes quantified by realtime RT-PCR. Blair P.L., et al. (2002) Nucleic Acids Research. 30(10): 2224-2231.
4. An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for
Hypersensitive PCR Applications. Champlot Sophie, Camille Berthelot, Mélanie Pruvost, E.
Andrew Bennett, Thierry Grange, Eva-Maria Geigl. (2010) PLoS ONE 5(9): e13042.
doi:10.1371/journal.pone.0013042.
5. Development of a novel rapid assay to assess the fidelity of DNA double-strand-break repair
in human tumour cells. Collis S.J., et al. (2002) Nucleic Acids Research. 30(2): e1.
6. Mutational analysis of the engrailed homeodomain recognition helix by phage display.
Connolly J., et al. (1999) Nucleic Acids Research. 27(4): 1182-1189.
AMRESCO, LLC Corporate Headquarters, 6681 Cochran Road Solon, OH 44139
Directions for Use
7. A novel method employing UNG to avoid carry-over contamination in RNA- PCR. Epstein
U.J., et al. (1993) Nucleic Acids Research. 21(16): 3917-3918.
8. Avoiding false positives with PCR. Kwok S., Higuchi R. (1989) Nature. 339: 237 – 238.
9. Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA
degradation and RNA amplification in reverse transcription-PCR. Kleiboeker S.B. (2005)
Virology Journal. 2: 29.
For Technical Support
Toll Free: 1-800-610-2789 (USA & Canada)
Fax: (440) 349-0235
Email: [email protected]
AMRESCO, LLC
A VWR Company
Corporate Headquarters
6681 Cochran Road
Solon, Ohio USA 44139-4300
Tel: 440/349-1199
Fax: 440/349-1182
www.amresco-inc.com
Uracil-DNA Glycosylase (UNG) Cod
ZY0615
Rev. 0 1/2015
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All Rights Reserved.
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AMRESCO, LLC Corporate Headquarters, 6681 Cochran Road Solon, OH 44139