Uracil-N-Glycosylase
This product is a recombinant UNG enzyme expressed and purified by Escherichia coli, with a protein molecular weight of 25kD. It can catalyze the release of free uracil from single and double stranded DNA containing uracil, and has no activity on RNA. It is mainly used to prevent contamination of PCR amplification products. The mechanism of action of this enzyme is to replace dTTP with dUTP in the PCR reaction, and all T in the amplified product fragments are replaced by U, forming PCR amplification products containing dU bases. UNG enzyme can selectively break the glycosidic bonds of U bases in single and double stranded DNA, degrade the DNA containing U in the reaction system, effectively eliminate residual contamination of PCR products, greatly reduce false positives caused by amplification product contamination, and ensure the specificity and accuracy of amplification.
Component 200U 1000U
Uracil-N-Glycosylase (1U/ μ L) 200 μ L 1mL
1 instruction manual and 1 copy
Storage: -20 ℃
The amount of enzyme required to catalyze 1 nmol of uracil from DNA containing uracil within 60 minutes at 37 ℃ is defined as one unit.
SDS-PAGE detection purity greater than 95%; No nucleic acid endonuclease or exonuclease activity was detected.
For long-term storage of UNG (not frequently used; less than 3 times a month), please store at -70 ℃, and for daily or weekly use, please store at -20 ℃. Try to avoid repeated freezing and thawing, and it is recommended to use separate packaging for large packages.
UNG can remove accidentally contaminated U-DNA molecules before PCR reactions, and a laboratory must use dUTP as one of the dNTPs in all PCR reactions, so that all amplification products become U-DNA. If used solely for a certain test, T-DNA will still accumulate, and this anti pollution system is also difficult to fully function.
The UNG/dUTP system is an internal pollution prevention measure for PCR reagents. In order to prevent contamination of PCR products, especially when repeatedly amplifying the same fragment in the clinical laboratory, strict laboratory division and operation must be standardized.
The following examples are the methods for using the Taq reaction system to prevent PCR product contamination. In practical applications, improvements and optimizations should be made based on specific experiments.
Prepare a PCR reaction system (using 50 μ L system as an example)
Component dosage and final concentration
ten × Taq PCR Buffer 5 μ L 1 ×
10mM dATP 1 μ L 200 μ M
10mM dGTP 1 μ L 200 μ M
10mM dCTP 1 μ L 200 μ M
10mM dTTP 0.5 μ L 100 μ M
10mM dUTP 1 μ L 200 μ M
Forward Primer (10 μ M) 1 μ L 0.2 μ M
Reverse Primer (10 μ M) 1 μ L 0.2 μ M
Template DNA X μ L-
Taq DNA Polymerase (5U/ μ L) 0.5 μ L 2.5U/50 μ L
Uracil-N-Glyco