Description
Addition of UNG in PCR, qPCR, and RT-PCR can prevent carryover contamination which leads to false positive results. Customers performing high-volume repetitive assays can benefit from the addition of UNG to their workflow. It is compatible with most PCR andRT-PCR reaction buffers, and active in a broad buffer range. This product is an better version compared to Thermolabile UDG (G5020L). Supplied in: 50 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol, pH 7.5 @ 25°C
Properties: Storage temperature: –25°C to –15°C Molecular weight: 51,727 Daltons Test Specifications SDS Purity >95% Single-stranded exonuclease <1.0% Double-stranded exonuclease <1.0% E. coli DNA contamination <10 copies
Uracil DNA Glycosylases (UDG) is a superfamily of enzymes (with 6 subfamilies) that act to repair DNA. Uracil-N-Glycosylase (UNG) is family 1 of the UDG superfamily, referring UNG to the gene. Both UDG and UNG perform the same biological function, removing uracil from DNA. Source of Protein: An E.coli strain carrying a cloned thermolabile uracil -N- glycosylase gene Unit Definition: 1 unit is defined as the amount of Thermolabile UNG 2.0 required to release 1nmol of uracil from dU-containing DNA in one hour at 37°C
Usage Instructions: Follow standard PCR assay guidelines Pipette gently Mix thoroughly Centrifuge the plate quickly before loading on instrument 2. Set up your quantitative PCR reaction per existing protocol. Thermolabile UNG (G5030-1000) is compatible with most PCR and RT-PCR reaction buffers. 3. Add 1µL Thermolabile UNG (G5030-1000) for each 50µL of master mix and vortex thoroughly. Table below is an example ofsetting up a one-step RT-qPCR reaction using a RNase H minus Reverse Transcriptase (such as Enzscript P7600L or StableScript P7720L) and an antibody-based hot-start Taq DNA polymerase (such as our Phoenix Hot Start Taq P7590L)). Step* °C Time Cycles UNG Treatment 25 2 min 1 Reverse transcription 50 10 min 1 RT inactivation and Initial Denaturation 94 3 min 1 Denaturation 94 15 sec 40 Annealing/Extension 60 60 sec *Optimal temperature and time for reverse transcription, RT inactivation and Taq activation, as well as Annealing/Extension condition, need to be determined by end user. Notes: Treatment of reaction with UNG for 2 minutes at 25°C at the beginning of thermal cycling removes uracil residues from dU-containing DNA, preventing it from serving as a template. Thermolabile UNG can be quickly heat-inactivated during reverse transcription at temperatures of at least 50°C for 10 minutes. Quality Control Analysis: Unit Activity i s measured using a 2-fold serial dilution method. Dilutions of the enzyme were made in 1X reaction buffer (70mM Tris-HCl, 10mM NaCl, 1mM 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, plunged on ice, and analyzed using a TCA-precipitation method. Protein Concentration (OD 280) 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 mass of the protein of interest band 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 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 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 16S rDNA Contamination is evaluated using 5 µL replicate samples of enzyme solution 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.
