Uracil N-glycosylase (UNG enzyme), also known as uracil DNA glycosylase (UDG enzyme), is a recombinant enzyme expressed and purified in E. coli. The protein has a molecular weight of 25 kD and can catalyze the release of free uracil from single and double stranded DNA containing uracil. It has no activity on RNA and 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 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
U665877 10000 U
Uracil-N-Glycosylase (UNG)，1 U/μL
10 mL
Unit Definition 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. Quality control SDS-PAGE detection purity greater than 95%; After testing, there was no activity of nucleic acid endonucleases or exonucleases. Matters needing attention 1. For long-term storage of UNG (infrequent use; less than 3 times per month), please store at -70 ℃. For daily or weekly use, please store at -20 ℃. Try to avoid repeated freeze-thaw cycles, and it is recommended to use separate packaging for large packages. 2. UNG can remove accidentally contaminated U-DNA molecules before PCR reactions. 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. 3. The UNG/dUTP system is an anti contamination measure inside PCR reagents. In order to prevent contamination of PCR products, especially when repeatedly amplifying the same fragment in the laboratory, strict laboratory division and operation must be standardized. Operation steps The following examples are the usage methods of Taq reaction system to prevent PCR product contamination. In practical applications, improvements and optimizations should be made based on specific experiments. 1. PCR reaction system
Reagent
50 μL Reaction system
Final concentration
Taq PCR Buffer，10×
5 μL
1 ×
dATP，10 mM
1 μL
200 μM
dGTP，10 mM
1 μL
200 μM
dCTP，10 mM
1 μL
200 μM
dTTP，10 mM
0.5 μL
100 μM
dUTP，10 mM
1 μL
200 μM
Forward Primer，10 μM
1 μL
0.2 μM
Reverse Primer，10 μM
1 μL
0.2 μM
Template DNA
X μL
/
Taq DNA Polymerase，5 U/μL
0.5 μL
2.5 U/50 μL
Uracil-N-Glycosylase (UNG)，1 U/ μL
0.2 μL
0.2 U/50 μL
ddH2O
up to 50 μL
/
2. Reaction conditions
Step
Temperature
Time
/
UNG digestion
37℃-50℃
5-10 min
/
Pre denaturation
95℃
10 min
/
Denaturation
94℃
30 s
30-40 cycles
Annealing
55-65℃
30 s
30-40 cycles
Extension
72℃
1 kb/min
30-40 cycles
Final extension
72℃
5 min
/
Note: Taq enzyme and UNG enzyme are usually added to the PCR reaction system in a certain proportion. They are first digested within the range of 37 ℃ -50 ℃ for 5-10 minutes, then inactivated at 95 ℃ for 10 minutes (this step also achieves pre denaturation and hot start effects), and then PCR amplification is carried out. The reaction of UNG enzyme can vary within the range of 37 ℃ -50 ℃, 5-10 minutes, and can be adjusted according to experimental needs.
