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Restriction Endonucleases >
Restriction Endonucleases >
HpyCH4III |
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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 HpyCH4III gene from Helicobacter pylori CH4 (S.A. Thompson).
Reagents Supplied:
NEBuffer 4
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 100% | | NEBuffer 2: | | 50% | | NEBuffer 3: | | 25% | | 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: Not sensitive |
Heat Inactivation:
80°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.13 unit(s)
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:
5,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
100 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
100 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent A
Notes
General notes:- Bst4CI is an isoschizomer of HpyCH4III.
- HpyCH4III produces DNA fragments that have been single base 3´ extension which are difficult to ligate.
- Overdigestions > 4 hours with > 5.0 units per µg DNA are not recommended.
FAQs
- Do degenerate recognition sites need to be palindromic?
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 5-fold overdigestion with HpyCH4III, approximately 50%
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 HpyCH4III.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 15 units of HpyCH4III 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.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA and 50units of enzyme incubated for 16 hours at 37ºC resulted in the same pattern of DNA bands as a reaction incubated for 1 hour with 1 unit of enzyme.
Reagents Sold Separately
NEBuffer 4
Legal
Patents:
New England Biolabs, Inc., Vanderbilt University: U.S. Patent No. 6,238,904
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