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Thermosensitive UDG enzyme
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Molecular Biological Enzymes
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Product Description
The thermosensitive UDG enzyme can hydrolyze the uracil glycosidic bond (base excision) of the DNA chain containing the U-DNA site to release uracil and produce the alkali-sensitive apyrimidinyl site (AP-DNA). This enzyme can act For single-stranded and double-stranded DNA containing uracil. , The
The thermosensitive UDG enzyme has lower activity on double-stranded DNA than single-stranded DNA. The enzyme is active on small U-DNA oligonucleotides and dUMP, but has no activity on RNA or normal non-uracil DNA. Since the thermosensitive UDG enzyme does not require metal ions, the enzyme is active in the presence of Mg2+ or EDTA.
Specification: 1 U/µL.
Applications
1. Cut DNA at the site where deoxyuridine acid residues have been incorporated. Through alkali treatment, high temperature or endonuclease (such as T4 endonuclease) specifically cleave the non-pyrimidine sites, which can catalyze the hydrolysis of AP-DNA;
2. Uracil-containing DNA (U-DNA) can be prepared in vitro. The thermosensitive UDG enzyme can be used to achieve site-specific, chain-specific or general cleavage, depending on how the U-DNA is prepared;
3. The enzyme can be used to improve the efficiency of targeted mutagenesis methods and to obtain highly labeled oligonucleotide probes;
4. The thermosensitive UDG enzyme can be used together with dUTP to eliminate PCR contamination of DNA synthesis reactions. In order to make the PCR product easy to degrade, dUTP must be substituted for dTTP in the PCR reaction mixture. Before PCR, the PCR reaction mixture must be pretreated with heat-sensitive UDG enzyme to degrade DNA containing uracil. Natural DNA does not contain uracil, so the sample will not be degraded.
Transportation and storage methods
Ice pack transportation. Store the product at -20°C.
Activity definition
The amount of thermosensitive UDG enzyme required to completely degrade 1 μg of purified single-stranded uracil DNA in 60 minutes at 37°C is defined as 1 activity unit (U).
Quality Inspection
Endonuclease residue detection: 10U of this enzyme and 0.6 μg λ DNA-Hind III digested product were incubated at 37°C for 16 hours, and the DNA electrophoresis band did not change.
RNase residue detection: 10U of this enzyme and 1 μg total RNA of HeLa cells were incubated at 37°C for 1 hour, and the electrophoretic band of RNA remained unchanged.
E.coli DNA residue detection: 10U of this product is tested by E.coli 16s rDNA specific TaqMan qPCR, and the E.coli genome residue is less than 10 copies.
Enzyme inactivation detection: 10U of this product is treated at 94°C for 2min, and incubated with 1μg of dU-containing dsDNA template for 30min, and the DNA electrophoresis band does not change.
Operation Manual
1. PCR reaction system (50 μL):
The thermosensitive UDG enzyme can hydrolyze the uracil glycosidic bond (base excision) of the DNA chain containing the U-DNA site to release uracil and produce the alkali-sensitive apyrimidinyl site (AP-DNA). This enzyme can act For single-stranded and double-stranded DNA containing uracil. , The
The thermosensitive UDG enzyme has lower activity on double-stranded DNA than single-stranded DNA. The enzyme is active on small U-DNA oligonucleotides and dUMP, but has no activity on RNA or normal non-uracil DNA. Since the thermosensitive UDG enzyme does not require metal ions, the enzyme is active in the presence of Mg2+ or EDTA.
Specification: 1 U/µL.
Applications
1. Cut DNA at the site where deoxyuridine acid residues have been incorporated. Through alkali treatment, high temperature or endonuclease (such as T4 endonuclease) specifically cleave the non-pyrimidine sites, which can catalyze the hydrolysis of AP-DNA;
2. Uracil-containing DNA (U-DNA) can be prepared in vitro. The thermosensitive UDG enzyme can be used to achieve site-specific, chain-specific or general cleavage, depending on how the U-DNA is prepared;
3. The enzyme can be used to improve the efficiency of targeted mutagenesis methods and to obtain highly labeled oligonucleotide probes;
4. The thermosensitive UDG enzyme can be used together with dUTP to eliminate PCR contamination of DNA synthesis reactions. In order to make the PCR product easy to degrade, dUTP must be substituted for dTTP in the PCR reaction mixture. Before PCR, the PCR reaction mixture must be pretreated with heat-sensitive UDG enzyme to degrade DNA containing uracil. Natural DNA does not contain uracil, so the sample will not be degraded.
Transportation and storage methods
Ice pack transportation. Store the product at -20°C.
Activity definition
The amount of thermosensitive UDG enzyme required to completely degrade 1 μg of purified single-stranded uracil DNA in 60 minutes at 37°C is defined as 1 activity unit (U).
Quality Inspection
Endonuclease residue detection: 10U of this enzyme and 0.6 μg λ DNA-Hind III digested product were incubated at 37°C for 16 hours, and the DNA electrophoresis band did not change.
RNase residue detection: 10U of this enzyme and 1 μg total RNA of HeLa cells were incubated at 37°C for 1 hour, and the electrophoretic band of RNA remained unchanged.
E.coli DNA residue detection: 10U of this product is tested by E.coli 16s rDNA specific TaqMan qPCR, and the E.coli genome residue is less than 10 copies.
Enzyme inactivation detection: 10U of this product is treated at 94°C for 2min, and incubated with 1μg of dU-containing dsDNA template for 30min, and the DNA electrophoresis band does not change.
Operation Manual
1. PCR reaction system (50 μL):
| Reagent | Sample volume |
| 10×PCR Buffer (Mg2+Plus) | 5μL |
| 25 mM MgCl2 | 3μL |
| dATP | 0.2 mM |
| dGTP | 0.2 mM |
| dCTP | 0.2 mM |
| dUTP | 0.8 mM |
| Primer 1（10 μM） | 2μL |
| Primer 1（10 μM） | 2μL |
| Taq DNA Polymerase（5 U/μL） | 0.5μL |
| Heat-Labile UDG（1 U/μL） | 1μL |
| ddH2O | up to 50 μL |
2. PCR reaction settings:
| Temperature reflex | Reaction time |
| 15～25℃ | 10 min |
| 94℃ | 2min |
| PCR reaction program |
Note: Heat inactivation: heating at 95°C for 2min. (If used for RT-PCR, heat at 55°C for 10 minutes for thermal inactivation.)
Precautions
1. For your safety and health, please wear lab coats and disposable gloves for operation.
Precautions
1. For your safety and health, please wear lab coats and disposable gloves for operation.
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