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FokI > FAQ |  | FokI FAQ
See the Restriction Endonucleases FAQ also.
Q1: Does FokI tend to degrade DNA?
Q2: Why does FokI degrade DNA?
Q3: Is extended digestion of FokI recommended?
Q4: Does FokI have trouble cleaving PCR products?
Q5: Is FokI blocked by methylation?
Q6: Is FokI used in special techniques?
Q7: What is the molecular weight of FokI?
Q8: Is FokI activity sensitive to pH?
Q9: Is FokI active at 25°C?
Q10: Does FokI cleave ssDNA?
Q1: Does FokI tend to degrade DNA?
A1: 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.
Q2: Why does FokI degrade DNA?
A2: 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.
Q3: Is extended digestion of FokI recommended?
A3: Extended digestion is not beneficial because the enzyme is not stable in reaction and it can degrade DNA.
Q4: Does FokI have trouble cleaving PCR products?
A4: Yes, the enzyme will totally degrade PCR products if too much enzyme is added.
Q5: Is FokI blocked by methylation?
A5: 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.
Q6: Is FokI used in special techniques?
A6: 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.
Q7: What is the molecular weight of FokI?
A7: 65.6 kD as determined from sequence data.
Q8: Is FokI activity sensitive to pH?
A8: The optimal pH range for FokI is between 7.0 and 8.5.
Q9: Is FokI active at 25°C?
A9: FokI exhibits 25-50% activity at 25°C.
Q10: Does FokI cleave ssDNA?
A10: No.
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