Description
Heat-labile UDG for carryover prevention
Heat-labile UDG is a uracil-DNA glycosylase that removes uracil from DNA to prevent carryover contamination in PCR and qPCR. It is then fully and irreversibly inactivated by brief heating, so it cannot degrade newly formed amplicons. It is ideal for contamination-sensitive workflows.
The enzyme is cloned from a psychrophilic marine bacterium and purified from E. coli. It hydrolyses the N-glycosidic bond of deoxyuridine to release uracil from single- and double-stranded DNA, removing aerosol contamination from dU-containing PCR products. Activity is irreversibly lost above 50°C. It is suitable for PCR, qPCR, RT-PCR and RT-qPCR and is compatible with most common PCR systems. For research use only.
Key Features
| Carryover prevention | Removes uracil from dU-containing DNA to eliminate contamination from previous PCRs. | |
| Rapid inactivation | Inactivated at 55°C for 10 min, and irreversibly above 50°C. | |
| Reliable compatibility | Works with a wide range of common PCR, qPCR, RT-PCR and RT-qPCR systems. | |
| High specific activity | Specific activity of ≥200,000 U/mg. |
Specifications
| Enzyme | Uracil-DNA glycosylase from a psychrophilic marine bacterium, purified from E. coli |
| Concentration | 1 U/μl |
| Specific activity | ≥200,000 U/mg |
| Inactivation | Irreversible above 50°C |
| Applications | PCR, qPCR, RT-PCR, RT-qPCR |
| Shelf life | 2 years |
Components
| Components | 100 U | 500 U |
| Heat-labile UDG (1 U/μl) | 100 μl | 500 μl |
Storage buffer: 20 mM Tris-HCl (pH 8.0 at 25°C), 0.1 mM EDTA, 100 mM KCl, 1 mM DTT, 50% glycerol (v/v), 0.5% NP-40 (v/v), 0.5% Tween-20 (v/v). Unit definition: one unit (U) releases 1 nmol of uracil from dU-containing DNA in 1 h at 37°C in 70 mM Tris-HCl (pH 7.5), 10 mM NaCl, 1 mM EDTA and 100 μg/ml BSA.
Storage
Store at -30 ~ -15°C.
- Prepare the PCR reaction mixture
- Set up a 50 μl reaction as follows:
| Components | Amount |
| ddH2O | To 50 μl |
| 10× Taq Buffer (with 20 mM MgCl2) | 5 μl |
| dUTPa | 0.6 mM |
| dATP/dCTP/dGTP | 0.2 mM each |
| Template DNA | Optional |
| Primer 1 (10 μM) | 2 μl |
| Primer 2 (10 μM) | 2 μl |
| Taq DNA Polymerase (5 U/μl) | 0.5 μl |
| Heat-labile UDG (1 U/μl)b | 1 μl |
a Adjust the final dUTP concentration to 0.2–0.6 mM as needed.
b Use 0.1–1 U of Heat-labile UDG per 50 μl reaction as needed.
The final Mg2+ concentration can be adjusted between 2 mM and 3 mM as needed.
- Run the PCR program
- Adjust the program as your experiment requires.
| Step | Temperature | Time | Cycles |
| Degradation of dU-containing templates | 25°C | 10 min | 1 |
| UDG inactivation and template denaturation | 95°C | 2 min | 1 |
| Denaturation | 94°C | 30 sec | 30–35 |
| Annealing | 55°C | 30 sec | 30–35 |
| Extension | 72°C | 60 sec/kb | 30–35 |
| Final extension | 72°C | 7 min | 1 |
Q1: How much Heat-labile UDG should I use?
- Use 0.1–1 U per 50 μl reaction, depending on your experiment; the standard protocol uses 1 μl (1 U).
Q2: How is the enzyme inactivated?
- It is heat-sensitive and irreversibly inactivated above 50°C. In the standard protocol, a 95°C step for 2 min inactivates the UDG and denatures the template.
Q3: Which reactions is it compatible with?
- It is suitable for PCR, qPCR, RT-PCR and RT-qPCR and works with most PCR and RT-PCR systems. Check compatibility with your own system before first use.
Q4: How much dUTP should be in the reaction?
- Use a final dUTP concentration of 0.2–0.6 mM, with dATP, dCTP and dGTP at 0.2 mM each.
