Thermo Scientific™ T7 Gene 6 Exonuclease

Description

• The T7 Gene 6 Exonuclease hydrolyzes duplex DNA non-processively in the 5'→3' direction from both 5'-phosphoryl or 5'-hydroxyl nucleotides by liberating oligonucleotides as well as mononucleotides, until about 50% of the DNA is acid soluble
• It also degrades nucleotides at the gaps and nicks of double-stranded DNA from the 5'-termini and RNA from RNA:DNA hybrids in the 5'→3' direction
• The T7 Gene 6 Exonuclease is similar to Lambda Exonuclease in that it catalyzes the stepwise hydrolysis of duplex DNA from the 5' termini liberating 5' mononucleotides
• However, unlike Lambda Exonuclease, the enzyme has low processivity and it will remove both 5'-hydroxyl and 5'-phosphoryl termini
• It also degrades RNA and DNA from RNA:DNA hybrids in the 5'→3' direction but is unable to degrade either double-stranded or single-stranded RNA
• This enzyme has been used for generating single-stranded DNA templates for sequencing or SNP analysis
• Molecular Weight: 32 kDa Optimum pH: 7.5 Optimum Temperature: 37 °C Inactivation: 75 °C for 10 min or add 2 μL of 0.5M EDTA for a 50 μL reaction volume
• Heating at 75 °C for 10 min or by adding 2 μL of 0.5M EDTA for a 50 μL reaction volume
• Greater than 95% pure as determined by SDS-PAGE
• Tested for contaminating endonucleases and ribonucleases
• The reaction mixture (50 μL) contains 50 mM Tris-HCI (pH 8.1), 5 mM MgCl 2 , 20 mM KCI, 5 mM 2-mercaptoethanol, double-stranded DNA, and enzyme
• Incubation is at 37°C for 15 min
• One unit is the amount of enzyme required to release 1 nmol of acid soluble nucleotide in 15 min at 37 °C under standard assay conditions
• 50 units/μL E
• coli strain containing an overproducing clone of the T7 Gene 6 Exonuclease Conversion of 0.5 pmol of λ DNA to single-stranded half molecules by 75 units of enzyme in 30 min at 37°C
• Verification by agarose gel electrophoresis
• Controlled stepwise digestion of double-stranded DNA from the 5' termini Generating ssDNA templates for sequencing or SNP analysis