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  • LpnPI

    This enzyme has transitioned to an improved new buffer system. Visit NEBCutSmart.com for further details.
     
    The new and current Double Digest Finder and current Activity/Performance Chart for the CutSmart buffer system are available. The previous version of the Double Digest Finder, as well as the previous Version of Activity/Performance Chart that use the former buffer system, are still available for your convenience.
    cloned at neb recombinant NEBuffer 4 incubation temp heat inactivation bsa
    R0663_v1_000017
    Catalog #SizeConcentrationPriceQtyAdd to Cart
    R0663S200 units5,000 units/ml$103.00Add to Cart
    R0663L1,000 units5,000 units/ml$412.00Add to Cart
      
    Categories:
    Epigenetic Analysis (EpiMark® Validated) Products,
    Methylation Dependent Restriction Enzymes,
    Restriction Endonucleases: H-M
    Applications:
    Methylated DNA Analysis,
    Restriction Enzyme Digestion,
    Restriction Enzymes for Epigenetics

    Description

    LpnPI, an EpiMark® validated product, is a modification-dependent endonuclease which recognizes CmCDG sites and generates a double-stranded DNA break on the 3´ side of the modified cytosine at N10/N14. Recognized cytosine modifications include C5-methylation (5-mC) and C5-hydroxymethylation (5-hmC) (1). 

    This enzyme is provided with an Enzyme Activator Solution which may be used for efficient digestion by LpnPI.

    The most common epigenetic modifications found in eukaryotic organisms are methylation marks at CpG or CHG sites. A subset of these modified sites are recognized and cleaved by LpnPI. 

    At fully methylated CpG sites: 
    5' . . . C mC  G G . . . 3'
    3' . . . G  G mC C . . . 5'

    or CHG sites: 
    5' . . . CmC D  G G . . . 3'
    3' . . . G G H mC C . . . 5'

    H = A or C or T (not G)
    D = A or G or T (not C) 

    LpnPI recognizes each hemi-methylated site individually and cleaves bidirectionally to generate 32-base or 31-base fragments, respectively. These fragments contain the central methylated site and have 4-base 5´ overhangs at each end. LpnPI does not cleave unmodified DNA.

    Product Source

    An E. coli strain that carries the synthetic LpnPI gene from Legionella pneumophila species Philadelphia 1.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 4-2010X
    Enzyme Activator Solution30X
    BSA-20100X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to digest 1 µg of pBR322 (dcm+) DNA in 1 hour at 37°C in a total reaction volume of 50 µl.

    Reaction Conditions

    1X NEBuffer 4
    Supplement with BSA
    Incubate at 37°C

    1X NEBuffer 4:
    50 mM Potassium Acetate
    20 mM Tris-acetate
    10 mM Magnesium Acetate
    1 mM DTT
    pH 7.9 @ 25°C

    Usage Concentration

    4,000 units/ml

    Activity in NEBuffers

    NEBuffer 1.1: 10%
    NEBuffer 2.1: 10%
    NEBuffer 3.1: 10%
    CutSmart™ Buffer: 50%

    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: Not Sensitive
    dcm methylation: Not Sensitive
    CpG Methylation: Not Sensitive

    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.
    • 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.
    • 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.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Notes

    1. Use of excess enzyme inhibits cleavage. Optimization of the amount of enzyme needed for complete digestion may be required for each substrate DNA.
    2. Star activity may result from extended digestion

    References

    1. Zheng, Y. et al. (2010). Nucl. Acids Res. doi:10, 1093/nar/gkq327.
    2. U.S. Publication No. 2010-0167942 Unpublished observation

    Supporting Documents

    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]

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
    2. How can I access the old NEBuffer Activity Chart?
    3. How can I access the old Double Digest Finder?
    1. Genomic DNA Digestion using LpnPI (R0663)
    2. Optimizing Restriction Endonuclease Reactions

    Selection Tools

    Usage Guidelines & Tips

    Troubleshooting Guides

    Interactive Tools