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Restriction Endonucleases >
Restriction Endonucleases >
HindIII |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
A E. coli strain that carries the HindIII gene from Haemophilus influenzae Rd (ATCC 51907).
Reagents Supplied:
NEBuffer 2
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 50% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 10% | | NEBuffer 4: | | 50% |
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: Not sensitive | |
More information about: Methylation Sensitivity |
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
(+ + +) Suitable for an extended or overnight digestion. Enzyme is active > 8 hours.
More information about: Extended Digests with Restriction Enzymes
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 2
Incubate at
37°C.
1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
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:
20,000 units/ml and 100,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
250 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
500 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent B
FAQs
- Will BSA affect HindIII activity?
- What is Star Activity and how can it be avoided?
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 200-fold overdigestion with HindIII, > 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 HindIII.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 400 units of HindIII incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. Note that the high enzyme concentration described in this assay may result in star activity.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 2,000 units of HindIII with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 40 units of HindIII with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
Blue/White Screening Assay:
An appropriate vector is digested at a unique site within the
lacZα gene with a 10-fold excess of enzyme.
The vector DNA is then ligated, transformed, and plated onto Xgal/IPTG/Amp plates.
Successful expression of β-galactosidase is a function of how intact its gene remains after cloning,
an intact gene gives rise to a blue colony, removal of even a single base gives rise to a white colony.
Enzyme preparations must produce fewer than 3% white colonies to be Blue/White certified.
HindIII crystals (Ira Schildkraut and Lydia Dorner, New England Biolabs)
Reagents Sold Separately
NEBuffer 2
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,180,673
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