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Restriction Endonucleases >
Restriction Endonucleases >
TfiI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
An E. coli strain that carries the cloned TfiI gene from Thermus filiformis (D. Cowan, University College London).
Reagents Supplied:
NEBuffer 4 (10X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 100% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 100% | | 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: Not sensitive | | CpG methylation: Blocked by some combinations of overlapping |
Activity at 37°C:
10%
Heat Inactivation:
No
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.25 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
Incubate at
65°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 65°C in a total reaction volume of 50 µl.
Concentration:
5,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
250 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
0.15% Triton X-100
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent C
Notes
General notes:- Conditions of low ionic strength, high enzyme concentration, glycerol concentration > 5%, or pH > 8.0 may result in star activity.
- Conditions of low ionic strangth, high enzyme concentration, glycerol concentration >5% or pH > 8.0 may result in star activity.
- TfiI can remain bound to DNA after cutting and alter migration rate of DNA during electrophoresis. To disrupt binding, add SDS to a final concentration of 0.5% or purify DNA before electrophoresis.
FAQs
- The NEB catalog has historically stated that activity in NEBuffer4 is less than 100% yet this enzyme is now supplied with NEBuffer4. What have you changed?
- Has the conversion to NEBuffer4 altered any of the properties of the restriction enzyme?
- If I have an old tube of enzyme, what NEBuffer should I use?
- Will the new enzyme work in the originally supplied NEBuffer?
- Why is NEB switching this restriction enzyme to NEBuffer 4?
- What is Star Activity and how can it be avoided?
- Do degenerate recognition sites need to be palindromic?
- How can this enzyme be inactivated?
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 TfiI, > 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, > 95% can be recut with TfiI.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 30 units of TfiI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 65ºC
released < 0.1% of the total radioactivity.
4-Hour Incubation:
A 50 µl reaction containing 1 µg of ϕ174 DNA and 5 units of TfiI incubated for 4 hours resulted in the same pattern of DNA bands as a reaction incubated for 1 hour with 1 unit of enzyme (see note).
Reagents Sold Separately
NEBuffer 4
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 6,133,008
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