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Restriction Endonucleases >
Restriction Endonucleases >
DpnII |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
A E. coli strain that carries the DpnII gene from Diplococcus pneumoniae G41 (S. Lacks).
Reagents Supplied:
NEBuffer DpnII (10X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | Not Recommended | | NEBuffer 2: | | Not Recommended | | NEBuffer 3: | | 100% | | NEBuffer 4: | | Not Recommended |
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: Blocked | | dcm methylation: Not sensitive | | CpG methylation: Not sensitive |
Heat Inactivation:
65°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 DpnII
Incubate at
37°C.
1X NEBuffer DpnII:
50 mM Bis-Tris-HCl
100 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 6.0 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA (dam-) in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration:
10,000 units/ml and 50,000 units/ml
Unit Assay Substrate:
λ DNA (dam-)
Storage Conditions:
10 mM Tris-HCl
200 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 B
Notes
Unlike other restriction enzymes that show Star Activity at high pH 8.0, DpnII has Star Activity above approximately pH 6.5. The effect of pH on Star Activity of DpnII is very extreme, showing one-thousand-fold more Star Activity at pH 7.5 than at pH 6.0. General notes:- Conditions of low ionic strength, high enzyme concentration, glycerol concentration > 5%, or pH > 8.0 may result in star activity.
- DpnII and Sau3AI are isoschizomers of MboI.
- Cleaves to leave a 5´ GATC extension which can be efficiently ligated to DNA fragments generated by BamHI, BclI, BglII, MboI, Sau3AI and BstYI.
- Blocked by dam methylation.
- DpnII is not recommended for use in any buffer except its own unique buffer because of the resultant lower activity and potential star activity.
- DpnII may be used in NEBuffer 3 for double digests but care should be taken to avoid star activity.
FAQs
- What's the difference between DpnI, DpnII, MboI, and Sau3AI?
- 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 50-fold overdigestion with DpnII, > 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 DpnII.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 100 units of DpnII 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 100 units of DpnII 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 30 units of DpnII 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.
Reagents Sold Separately
NEBuffer DpnII
Companion Products
dam-/dcm- Competent E. coli
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