New England Biolabs, R0109S, FokI

FokI has been reformulated, and now also includes Recombinant Albumin (rAlbumin) beginning with Lot #10295609. Related Categories Restriction Endonucleases C G Applications Restriction Enzyme Digestion Specification Unit Definition One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 μl. Reaction Conditions 1X rCutSmart™ Buffer Incubate at 37°C 1X rCutSmart™ Buffer 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Activity in NEBuffers NEBuffer™ r1.1: 100% NEBuffer™ r2.1: 100% NEBuffer™ r3.1: 75% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent A Storage Buffer 10 mM Tris-HCl 50 mM NaCl 1 mM DTT 0.1 mM EDTA 200 µg/ml Recombinant Albumin 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Impaired by Overlapping CpG Methylation: Impaired by Overlapping FAQ Q: Does FokI tend to degrade DNA? A: Yes. Use a maximum of 2-3 units/μg DNA, for 1 hour or less. Tween 20 can be added to a final concentration of 0.1% to reduce degradation. Q: Why does FokI degrade DNA? A: FokI has two domains; one that recognizes DNA and one that cleaves. The protein is unstable and as the specific activity is lost an increasing nonspecific activity results. Tween 20 stabilizes the protein and slows the increase in nonspecific activity. Q: Is extended digestion of FokI recommended? A: Extended digestion is not beneficial because the enzyme is not stable in reaction and it can degrade DNA. Q: Is FokI blocked by methylation? A: FokI will cut 2-4 times slower if the end C in the Sequence is followed by a G and is methylated. Also, FokI will not cut DNA comletely if there is an MspI methylation site before the recognition site. For up-to-date information about methylation sensitivities, please visit Dam-Dcm and CpG Methylation or REBASE. Q: Is FokI used in special techniques? A: FokI is used to cleave between any two base pairs. See Podhajska, A.J., Szybalski, W. (1985). Gene 40: 175-182 In brief, you synthesize an oligonucleotide which contains a double-stranded FokI site and a single-stranded extension which anneals to the DNA template at the point where you would like to cut. Anneal the oligonucleotide duplex to the single stranded DNA template. In the subsequent cleavage reaction, the FokI will bind to the double-stranded site on teh oligonucleotide duplex, and will cut the template to which it is annealed. Q: What is the molecular weight of FokI? A: 65.6 kD as determined from sequence data. Q: Is FokI activity sensitive to pH? A: The optimal pH range for FokI is between 7.0 and 8.5. Q: Is FokI active at 25°C? A: FokI exhibits 25-50% activity at 25°C. Q: Does FokI cleave ssDNA? A: No. Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price.