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                    Products > Virus-related Products > Heat Labile UDG R5001

                    Heat Labile UDG R5001

                    Heat Labile UDG

                    For research use only

                    Components  

                    Component

                    R5001 (500 U)

                    Heat Labile UDG (1 U/μl)

                    500 μl 

                     

                    Storage

                    This reagent should be kept at -30~15°C.

                     

                    Description

                    Heat Labile UDG is purified from the expression product of an recombinant E. coli strain containing the UDG gene cloned from cold-adaptive marine bacteria (psychrophilic Marine bacterium). UDG (Uracil-DNA Glycosylase) catalyzes the hydrolysis of uracil bases of the single or double stranded DNA containing dU and the N-glycoside bonds of the glycosphosphoric acid skeleton to release free uracil. The resulting base-free site is easily fractured by hydrolysis. This product is sensitive to high temperature, and above 50°C can make the enzyme irreversible inactivation, suitable for PCR, qPCR, RT-PCR, RT-qPCR system.

                     

                    Storage Buffer

                    20 mM Tris-HCl, pH 8.0 at 25°C

                    0.1 mM EDTA

                    100 mM KCl

                    1 mM DTT

                    Glycerol 50% (v/v)

                    NP - 40 0.5% (v/v)

                    0.5% Tween - 20 (v/v)

                     

                    Unit Definition

                    In 70 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM EDTA, 100 g/ml BSA reaction solution, the amount of enzyme required to release 1 nmol uracil from DNA containing dU within 1 h at 37°C was defined as 1 unit of activity (U).

                     

                    Specific Activity

                    ≥200,000 U/mg

                     

                    Quality Control

                    Exonuclease residue detection: 20 U of this product and 0.3 μg of λ-Hind III were incubated at 37°C for 16 h, and the DNA bands did not change after electrophoresis.

                    Detection of endonuclease residue: 20 U of this product and 0.3 μg of Supercoiled pBR322 DNA were incubated at 37°C for 4 h, and the DNA bands did not change after electrophoresis.

                    RNase residue detection: the product of 20 U and 1 μg of HeLa cell RNA were incubated at 37°C for 30 min, and the electrophoresis band of RNA was unchanged by agarose gel electrophoresis.

                    E.coli DNA residue detection: the nucleic acid residue in 20 U of this product was detected by E. coli gDNA-specific TaqMan qPCR, and the residue of E. coli genome was less than 10 copies.

                     

                    Protocol

                    1. Prepare the PCR reaction system as follows:

                    Component

                    Amount

                    10 × Taq Buffer (Mg2+ plus)

                    5 μl

                    dUTP Mixa

                    0.6 mM

                    dATP/dCTP/dGTP

                    0.2 mM each

                    Primer 1 (10 μM)

                    2 μl

                    Primer 2 (10 μM)

                    2 μl

                    Template DNA

                    x μl

                    Taq DNA Polymerase (5 U/μl)

                    0.5 μl

                    Heat Labile UDG (1 U/μl)b

                    1 μl

                    ddH2O

                    to 50 μl

                    a. According to the needs of the experiment, the final dUTP concentration can be adjusted between 0.2-0.6 mM.

                    b. According to the needs of the experiment, the amount of 50 μl reaction system is generally 0.1-1 U.

                    * According to the experimental needs, the final concentration of MgCl2 can be adjusted between 2-3 mM.

                     

                    2. Reaction procedure

                    Temperature

                    Time

                    Description

                    25°C

                    10 min

                    degradation of U - containing templates

                    95°C

                    2 min

                    UDG inactivation, template denaturation

                    94°C

                    30 sec

                    30~35 cycles

                    55°C*

                    30 sec

                    72°C

                    60 sec/kb

                    72°C

                    7 min

                    final extension

                    * The PCR reaction procedure can be adjusted according to the experimental needs. 


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