DpnII

recombinant timesaver 5min unique buffer dil_B-grey incubation temp heat inactivation dam
Dpn-II-cutsite_1
Catalog #SizeConcentrationPriceQtyAdd to Cart
R0543S1,000 units10,000 units/ml$68.00Add to Cart
R0543T1,000 units50,000 units/ml$68.00Add to Cart
R0543L5,000 units10,000 units/ml$275.00Add to Cart
R0543M5,000 units50,000 units/ml$275.00Add to Cart
  
Categories:
Epigenetic Analysis (EpiMark® Validated) Products,
Methylation Sensitive Restriction Enzymes for Epigenetics,
Restriction Endonucleases: C-G
Applications:
Restriction Enzyme Digestion,
Restriction Enzymes for Epigenetics

Description

Product Source

An E. coli strain that carries the DpnII gene from Diplococcus pneumoniae G41 (S. Lacks).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
NEBuffer DpnII-2010X

Properties and Usage

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.

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 DTT
pH 6 @ 25°C

Activity in NEBuffers

NEBuffer 1.1: 25%
NEBuffer 2.1: 25%
NEBuffer 3.1: 100%
CutSmart® Buffer: 25%

Diluent Compatibility

Storage Temperature

-20°C

Storage Conditions

10 mM Tris-HCl
300 mM NaCl
1 mM DTT
0.1 mM EDTA
500 μg/ml BSA
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 min

Methylation Sensitivity

dam methylation: Blocked
dcm methylation: Not Sensitive
CpG Methylation: Not Sensitive

Notes

  1. DpnII and Sau3AI are isoschizomers of MboI.
  2. Cleaves to leave a 5´ GATC extension which can be efficiently ligated to DNA fragments generated by BamHI, BclI, BglII, MboI, Sau3AI and BstYI.
  3. This enzyme is blocked by dam methylation. More information can be found at Dam-Dcm and CpG Methylation.
  4. Will exhibit star activity in NEBuffer 3.1. We recommend the use of NEB DpnII Unique Buffer.

FAQs

  1. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
  2. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
  3. How can I access the old NEBuffer Activity Chart?
  4. Why is my Restriction Enzyme not cutting DNA?
  5. Why do I see a DNA smear on an agarose gel after a restriction digest?
  6. Why do I see additional DNA bands on my gel after a restriction digest?
  7. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?

Tech Tips

This enzyme will not cut plasmids isolated from most E.coli because it is blocked by dam methylation. For enzyme cleavage to occur, plasmids must be transformed and isolated from a dam-, dcm- strain of E.coli, such as NEB’s dam-/dcm- Competent E.coli (NEB #C2925).

Protocols

  1. Optimizing Restriction Endonuclease Reactions
  2. Double Digest Protocol with Standard Restriction Enzymes
  3. Time-Saver Protocol for Restriction Enzyme Digests
  4. Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)
  5. Protocol for Digestion Prior to droplet digital PCR (ddPCR)

Selection Charts

Usage Guidelines & Tips

Troubleshooting Guides

Interactive Tools

Citations

  • Hughes J.R., Roberts N., McGowan S., Hay D., Giannoulatou E., Lynch M., De Gobbi M., Taylor S., Gibbons R., Higgs D.R. (2014). Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment Nat Genet. 46 (2), 205-212. PubMedID: 24413732, DOI: doi:10.1038/ng.2871

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Endonuclease Activity (Nicking):
    The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
  • Exonuclease Activity (Radioactivity Release):
    The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
  • Ligation and Recutting (Terminal Integrity):
    After an over-digestion of DNA with a restriction endonuclease the percentage of the DNA fragments ligated with T4 DNA ligase and the percentage that can be recut are determined by agarose gel electrophoresis.
  • Non-Specific DNase Activity (16 hour):
    The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.