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| Product Name | Uracil-DNA Glycosylase (UDG), Heat-Labile, 1 U/uL, Recombinant |
| Catalog No. | IEC-HMM-0080 |
| Description | Heat-labile recombinant uracil-DNA glycosylase (UDG, also known as uracil-N-glycosylase or UNG) from a psychrophilic marine bacterium, engineered for complete and irreversible heat inactivation at 50 C for 10 minutes. UDG catalyzes the hydrolysis of the N-glycosidic bond between uracil and the deoxyribose sugar in single-stranded and double-stranded DNA containing uracil residues (from dUTP incorporation or cytosine deamination), generating abasic (AP) sites. This enzyme is essential for carryover prevention in PCR using dUTP instead of dTTP — UDG treatment of PCR setup mixtures degrades any uracil-containing contaminant amplicons from previous reactions while leaving the natural thymine-containing template DNA intact. The heat-labile property ensures complete inactivation before PCR cycling begins, preventing degradation of newly synthesized uracil-containing PCR products. Also used in site-directed mutagenesis and DNA repair research. |
| Intended Use | Carryover prevention in PCR by eliminating uracil-containing contaminating DNA from previous amplifications (dUTP/UDG system); site-directed mutagenesis (uracil-containing single-stranded DNA template degradation); DNA repair enzyme activity studies; preparation of abasic site-containing DNA substrates for biochemical assays; removal of uracil from DNA prior to bisulfite conversion for methylation analysis. |
| Principle / Technology | UDG scans DNA and flips out uracil bases from the DNA helix, cleaving the N-glycosidic bond between uracil and deoxyribose, releasing free uracil and leaving an abasic (AP) site. The enzyme acts on both single-stranded and double-stranded DNA with preferential activity on ssDNA. Heat-labile variant from psychrophilic organism denatures completely and irreversibly at 50 C (10 min), well below standard PCR denaturation temperatures. In dUTP/UDG carryover prevention: dUTP replaces dTTP in PCR, generating uracil-containing amplicons; subsequent PCR reactions are pre-treated with UDG, which degrades any contaminating uracil-DNA from previous reactions but spares natural thymine-containing template DNA. |
| Detection Method | Add 0.5-1 U UDG per 50 uL PCR reaction; incubate 10 min at 25-37 C (uracil-DNA degradation); heat-inactivate at 50 C for 10 min (enzyme inactivation + AP site cleavage by heat); proceed with standard PCR thermal cycling. |
| Sample Type | PCR reaction mixture before addition of DNA polymerase and template (for carryover prevention); purified DNA substrates (for in vitro biochemical assays). |
| Performance Range / Specifications | Activity: 1 U/uL (1 U = amount of enzyme that releases 1 nmol uracil per minute from uracil-containing single-stranded DNA at 37 C); heat inactivation: >99.9% inactivation at 50 C for 10 min; carryover prevention: degrades up to 10^7 copies of uracil-containing contaminant DNA in a standard 50 uL PCR reaction. |
| Sensitivity / LOD | Detection and removal of as few as 10 copies of uracil-containing contaminant DNA by qPCR monitoring; effective carryover elimination in PCR with as few as 0.2 U per 50 uL reaction. |
| Specificity | UDG specifically cleaves uracil from DNA (both ssDNA and dsDNA); does not recognize thymine (5-methyluracil), cytosine, 5-methylcytosine, adenine, or guanine; does not cleave uracil from RNA; minor activity on 5-fluorouracil in DNA; the heat-labile variant shows no detectable activity after 50 C inactivation. |
| Reaction Conditions / Protocol | UDG treatment: 10 min at 25-37 C (optimal 37 C) in standard PCR buffer; heat inactivation: 10 min at 50 C; typical PCR setup: add UDG to master mix, incubate 10 min at 25 C (carryover degradation), 50 C 10 min (inactivation), then standard cycling from 95 C. |
| Components / Formulation | Uracil-DNA Glycosylase, Heat-Labile (1 U/uL, in 20 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 50% glycerol), 10x UDG Reaction Buffer (200 mM Tris-HCl pH 8.0, 10 mM DTT, 10 mM EDTA), Protocol. |
| Storage Conditions | Store at -20 C; avoid repeated freeze-thaw cycles. |
| Shelf Life | 24 months from date of manufacture at -20 C. |
| Package Specifications | 100 U (100 uL), 500 U (500 uL), 2,500 U (2.5 mL). |
| Product Form | Liquid; 1 U/uL in 50% glycerol storage buffer. |
| Quality Control | Each lot tested: uracil release activity from tritiated uracil-DNA substrate; heat lability: <0.1% residual activity after 50 C for 10 min; no detectable non-specific endonuclease or exonuclease activity on supercoiled plasmid DNA; no detectable DNase or RNase contamination; PCR carryover prevention verified: complete elimination of 10^7 copies uracil-containing amplicon without affecting thymine template amplification. |
| Key Features | Heat-labile (complete inactivation at 50 C, 10 min); no interference with downstream PCR; carryover prevention with dUTP system; effective on ssDNA and dsDNA; does not affect natural template DNA; compatible with all standard PCR buffers. |
| Purity | >95% pure by SDS-PAGE; no detectable DNase, RNase, endonuclease, or exonuclease activities. |
| Concentration | 1 U/uL; protein concentration approximately 0.1-0.5 mg/mL. |
| Activity / Unit Definition | 1 U = amount of enzyme that catalyzes the release of 1 nmol uracil per minute from tritiated uracil-containing single-stranded DNA at 37 C in 1x reaction buffer. |
| Molecular Weight | Approximately 25-28 kDa (monomer, depending on source organism). |
| Source / Origin | Recombinant UDG from a psychrophilic marine bacterium (e.g., Psychrobacter sp. or similar cold-adapted organism), expressed in E. coli; purified by affinity and ion exchange chromatography; no animal-derived components. |
| pH Range / Optimal pH | Optimal pH 7.5-8.5; active range pH 6.0-9.5; 10x UDG Reaction Buffer pH 8.0. |
| Shipping Conditions | Cold pack or dry ice; stable at ambient for up to 3 days. |
| Expiration Date / Stability | 24 months at -20 C; quality retained after 10 freeze-thaw cycles; avoid storage at temperatures above -20 C for extended periods. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. ISO 9001 certified manufacturing. |
| Compatibility | Compatible with all standard PCR buffers (Tris-based, Mg2+-containing). Addition of 0.5-1 U UDG per 50 uL PCR reaction does not inhibit Taq or high-fidelity DNA polymerases. Compatible with dNTP mixes containing dUTP (typically dTTP replaced partially or fully with dUTP). The UDG pre-treatment step (25-37 C, 10 min) must be added before the initial denaturation step. Heat inactivation at 50 C for 10 min is compatible with most hot-start polymerases (antibody-mediated or chemical modification) as they remain inactive at this temperature. For RT-PCR applications, note that UDG may degrade uracil-containing cDNA from RNA templates — use standard dTTP in RT step and dUTP only in PCR step. |
| Recommended Buffer System | 10x UDG Reaction Buffer: 200 mM Tris-HCl pH 8.0, 10 mM DTT, 10 mM EDTA; enzyme storage: 20 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 50% glycerol. |
| Application Notes / Precautions | Include 0.2-0.4 mM dUTP in PCR instead of dTTP for the dUTP/UDG carryover prevention system. The UDG pre-treatment step should be integrated into the PCR program: 25 C for 10 min (UDG digest), 50 C for 10 min (UDG inactivation + AP site hydrolysis), then 95 C initial denaturation and standard cycling. If separate dNTP stocks are used, prepare dNTP-dUTP mix with dATP, dCTP, dGTP (0.2 mM each) and dUTP (0.4 mM) — the higher dUTP compensates for slightly lower incorporation efficiency by Taq polymerase. PCR products containing dUTP should be stored at -20 C and can be used for downstream cloning (restriction enzymes and T4 DNA ligase function normally with dUTP-substituted DNA). Do not use UDG-treated PCR products for applications requiring intact uracil residues. |
| Batch-to-Batch Consistency | Activity within 1.0 +/-0.15 U/uL for all lots; heat inactivation >99.9% at 50 C for 10 min; PCR carryover prevention efficacy verified for each lot. |
For research use only, not for clinical use.
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