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Restriction Endonucleases >
Restriction Endonucleases >
FokI |
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Prices are in US dollars and valid only for US orders.
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
A E. coli strain that carries the FokI gene from Flavobacterium okeanokoites (IFO 12536).
Reagents Supplied:
NEBuffer 4
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 100% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 75% | | NEBuffer 4: | | 100% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Methylation Sensitivity:
| dam methylation: Not sensitive | | dcm methylation: Impaired by overlapping | | CpG methylation: Impaired by overlapping | |
More information about: Methylation Sensitivity |
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
(–) Not recommended for digest over 1 hour.
More information about: Extended Digests with Restriction Enzymes
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
Incubate at
37°C.
1X NEBuffer 4:
20 mM Tris-acetate
50 mM potassium acetate
10 mM Magnesium Acetate
1 mM Dithiothreitol
pH 7.9 @ 25°C
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.
Concentration:
4,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
50 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent A
Notes
General notes:- FokI can cleave between virtually any two nucleotides by constructing a complementary oligonucleotide to the sequence to be cleaved (Szybalski, W. (1985) Gene 40, 169-173, Podhajska, A. and Szybalski, W. (1985) Gene 40, 175-182).
- Overdigestions of >5 units of FokI per μg of DNA and incubation times >2 hours are not recommended.
FAQs
- Does FokI tend to degrade DNA?
- Why does FokI degrade DNA?
- Is extended digestion of FokI recommended?
- Does FokI have trouble cleaving PCR products?
- Is FokI blocked by methylation?
- Is FokI used in special techniques?
- What is the molecular weight of FokI?
- Is FokI activity sensitive to pH?
- Is FokI active at 25°C?
- Does FokI cleave ssDNA?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Ligation and Re-cutting:
After a 10-fold overdigestion with FokI, > 95%
of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM)
at 16ºC. Of these ligated fragments, approximately 75% can be recut with FokI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 4 units of FokI incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 6 units of FokI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.3% of the total radioactivity.
FokI crystals (Ira Schildkraut and Lydia Dorner, New England Biolabs)
Reagents Sold Separately
NEBuffer 4
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 4,999,294
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