Cat. No. / ID: G5020L
Thermolabile UDG removes uracil from DNA by hydrolyzing the N-glycosylic bond between the deoxyribose and the base leaving an apurinic or apyrimidinic site. This enzyme (1–10 units) is completely inactivated by a 10-minute incubation at temperatures greater than 50°C in the 1x reaction buffer as measured in the unit characterization assay.
This enzyme is supplied in 50 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 7.5 at 25°C.
10X Reaction Buffer (cat. no. B5020) contains the following: 700 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA and 1 mg/ml BSA; pH 8.0 at 25°C.
Ask about low glycerol options
SDS available on request.
|Specific activity||n/a||30,000 U/mg|
|Single-stranded exonuclease||10 units||<1.0% released|
|Double-stranded exonuclease||10 units||<1.0% released|
|Double-stranded endonuclease||5 units||No conversion|
|E. coli DNA contamination||5 units||<10 copies|
Source of recombinant enzyme protein
The protein is produced by a recombinant E. coli strain carrying the recombinant thermolabile uracil DNA glycosylase gene isolated from a marine bacteria.
Unit definition: One unit is defined as the amount of Thermolabile UDG required to release 1 nmol of uracil from dU-containing DNA in one hour at 37°C.
Customers performing high-volume, repetitive assays can benefit from addition of Uracil-DNA Glycosylase (UDG) to their workflow to exclude carryover contamination.
PCR experiments are performed using dUTP in the nucleotide mix and incubated with UDG prior to the PCR amplification. UDG removes uracil from the PCR product so that, in each new reaction, residual products are digested and prevented from amplifying while the actual DNA templates are unaffected. This prevents carryover contamination from PCR, qPCR and RT-PCR that leads to false positive results.
Previously, cDNA synthesis temperatures were limited by the optimal reaction temperatures of earlier generation reverse transcriptases.Low-temperature set up at 50°C of the cDNA synthsis step causes delayed Cq values because the newly amplified DNA containing dUTP is cleaved by UDG. Users must choose between preventing carryover contamination and delaying Cq or risking carryover contamination and maintaining Cq values.
Using an elevated cDNA synthesis step at 55°C maintains Cq values. QIAGEN OEM portfolio includes StableScript Reverse Transcriptase (cat. no. P7720L), a thermostable M-MLV RNase H minus reverse transcriptase. The StableScript enzyme has an optimal reaction temperature of 50°C to 65°C and pairs well with QIAGEN’s Thermolabile UDG (cat. no. G5020L).
Quality control analysis
Unit activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer (70 mM Tris-HCl, 10 mM NaCl, 1 mM EDTA, 100 µg/ml BSA: pH 8.0 @ 25°C) and added to reactions containing a 3H-dUTP labeled 1.1kb PCR product in 1X reaction buffer. Reactions were incubated for 60 minutes at 37°C, placed on ice, and analyzed using a TCA-precipitation method.
Protein concentration is determined by OD280 absorbance.
Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.
Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.
Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.
Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.
E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.
Thermolability was examined by heat treating 10 units of enzyme for 10 minutes at 50°C in 1X reaction buffer. The enzyme was transferred to reactions containing a 3H-dUTP 1.1kb PCR product and activity was measured as described in the unit characterization assay. After heat treatment, less than 0.5% activity remains.
This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.