LpnPI

As of October 1, 2015, this product is available as a small size only. For larger volume purchases, please contact us.
cloned at neb recombinant incubation temp heat inactivation EpiMark Icon
R0663_v1_000017
Catalog #SizeConcentrationPriceQtyAdd to Cart
R0663S200 units5,000 units/ml$106.00Add to Cart
  
Categories:
Epigenetic Analysis (EpiMark® Validated) Products,
Methylation Dependent Restriction Enzymes for Epigenetics,
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
CutSmart® Buffer-2010X
Enzyme Activator Solution30X

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 CutSmart® Buffer
Supplement with 1X Enzyme Activator Solution
Incubate at 37°C

1X CutSmart® Buffer:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 μg/ml BSA
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: 100%

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

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. Excess of enzyme or prolonged digestion time in the presence of Enzyme Activator Solution may cause star activity.
  2. Based on the stability of the enzyme in the reaction, incubations longer than 1 hr will not result in improved digestion, unless additional enzyme is added. Please refer to Restriction endonuclease survival in a reaction for more information regarding this topic.
  3. 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

FAQs

  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. Why is my Restriction Enzyme not cutting DNA?
  4. Why do I see a DNA smear on an agarose gel after a restriction digest?
  5. Why do I see additional DNA bands on my gel after a restriction digest?
  6. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?

Protocols

  1. Genomic DNA Digestion using LpnPI (R0663)
  2. Optimizing Restriction Endonuclease Reactions
  3. Double Digest Protocol with Standard Restriction Enzymes

Selection Charts

Usage Guidelines & Tips

Troubleshooting Guides

Interactive Tools

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.
  • 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 (2 Hour Digestion):
    The product is tested in a reaction containing a RNA substrate.  After incubation for 2 hour there is no detectable degradation of the RNA substrate as determined by 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.