New England Biolabs, M0240S, Fpg

Related Categories DNA Repair Enzymes and Structure-specific Endonucleases Applications Polymerases for DNA Manipulation Specification Unit Definition One unit is defined as the amount of enzyme required to cleave 10 pmol of a 34-mer oligonucleotide duplex containing a single 8-oxoguanine base paired with a cytosine in a total reaction volume of 10 μl in 1 hour at 37°C. Reaction Conditions 1X NEBuffer™ 1 Supplement with 100 µg/ml Recombinant Albumin, Molecular Biology Grade Incubate at 37°C 1X NEBuffer™ 1 10 mM Bis-Tris-Propane-HCl 10 mM MgCl2 1 mM DTT (pH 7 @ 25°C) Dilution for Comet Assay Recommended dilution for the Comet Assay: 1:10 3 to 1:10 4 (4,5,6). For a protocol please visit: http://cometassay.com . Storage Buffer 20 mM Tris-HCl 50 mM NaCl 0.5 mM EDTA 200 µg/ml BSA 50% Glycerol pH 8 @ 25°C Heat Inactivation 60°C for 10 minutes FAQ Q: What is the activity of Fpg in the NEBuffers 1-4? A: Fpg has 100% activity in NEBuffers 1, 2 and 4. It has 75% activity in NEBuffer 3. Q: Does Fpg cleave hydroxymethyl uracil? A: No. Fpg will not cleave hydroxymethyluracil. Q: Does Fpg cut only one strand, or do they cause a double-strand break? A: Fpg cuts the strand that contains the damage. If there is damage on both strands, then it will cut both. Q: What is the Molecular Weight of Fpg? A: The molecular weight of Fpg is 30 kDa. Q: What is the difference between using Fpg and hOOG1? A: hOGG1 cuts the bond 3' to the modified base. This leaves a nick and the modified base in the DNA substrate. Fpg has the same basic substrate specificity, but it cuts 5' and 3' to the modified base. The modified base floats away and the DNA is left with a gap where the modified base used to be. Q: Does the Fpg contain a tag? A: The Fpg protein that NEB sells does not have a tag. Q: What buffer will work with both Endonuclease III and Fpg? A: Any NEBuffer 1-4 will work for both enzymes. Q: What substrate is Fpg tested on? A: The substrate used to test Fpg is described in the unit definition. It is a 34-mer oligonucleotide duplex containing a single 8-oxoguanine base paired with a cytosine. Q: What is the activity of Fpg in rCutSmart Buffer? A: Fpg is 100% activity in rCutSmart Buffer. Please refer to Activity of DNA Modifying Enzymes in rCutSmart™ Buffer for the activity of other enzymes in rCutSmart Buffer.