Description
Thermolabile UDG removes uracil from DNA by hydrolyzing the N-glycosylic bond between the deoxyribose and the base, leaving an apurinic or apyrimidinic site. There is no activity shown on RNA substrates, making it usable for RT-PCR. 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. The 10x Reaction Buffer contains 700 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA and 1 mg/mL BSA; pH 8 at 25°C.
Storage temperature: –25°C to –15°C Molecular weight: 26,218 Daltons Test Amount tested Specification Purity n/a >98% Specific activity n/a 30,000 U/mg Single-stranded exonuclease 10 units <1% released Double-stranded exonuclease 10 units <1% released Double-stranded endonuclease 5 units No conversion E. coli DNA contamination 5 units <10 copies
The protein is produced by a recombinant E. coli strain carrying the recombinant thermolabile uracil DNA glycosylase gene isolated from a marine bacteria. One unit is the amount of Thermolabile UDG required to release 1 nmol of uracil from dU-containing DNA in one hour at 37°C. Notes: If performing high-volume, repetitive assays can benefit from adding 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 before 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 setup at 50°C of the cDNA synthesis step causes delayed C q values because the newly amplified DNA containing dUTP is cleaved by UDG. Users must choose between preventing carryover contamination, delaying C q, risking carryover contamination, and maintaining C q values. Using an elevated cDNA synthesis step at 55°C maintains C q values. The StableScript enzyme has an optimal reaction temperature of 50–65°C and pairs well with Thermolabile UDG (G5020L).
Usage Instructions Use the included UDG reaction buffer at 1x. This enzyme is active in most molecular biology reaction buffers, so there is no need to exchange buffers. Add 0.5 µL UDG (G5020L) to a reaction with uracil-containing DNA substrate (up to 0.1 µg) in a 20 µL total reaction. Incubate at 25–37°C for 30 minutes. This enzyme can be completely inactivated by a 10-minute incubation at temperatures greater than 50°C. Note: Protocol can be modified for application-specific usage. Quality Control Unit activity was measured using a twofold serial dilution method. Dilutions of the 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 OD 280 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 band's mass 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.
