PflMI

Description

Product Source

An E. coli strain that carries the PflMI gene from Pseudomonas fluorescens (R. Morgan).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
NEBuffer 3.1-2010X

Properties and Usage

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.

Reaction Conditions

1X NEBuffer 3.1
Incubate at 37°C

1X NEBuffer 3.1:
100 mM NaCl
50 mM Tris-HCl
10 mM MgCl2
100 μg/ml BSA
pH 7.9 @ 25°C

Activity in NEBuffers

NEBuffer 1.1: 0%
NEBuffer 2.1: 100%
NEBuffer 3.1: 100%
CutSmart® Buffer: 50%

Diluent Compatibility

Storage Temperature

-20°C

Storage Conditions

10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
200 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: Blocked by Overlapping
CpG Methylation: Not Sensitive

Notes

  1. Particular PflMI sites in λ DNA are cleaved at significantly lower rates than those found in other substrates.
  2. Star activity may result from a glycerol concentration of >5%

FAQs

  1. Do degenerate recognition sites need to be palindromic?
  2. My restriction enzyme used to be available at a lower concentration. Why does it now come at a higher concentration of 10,000 u/ml?
  3. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
  4. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
  5. How can I access the old NEBuffer Activity Chart?
  6. Why is my Restriction Enzyme not cutting DNA?
  7. Why do I see a DNA smear on an agarose gel after a restriction digest?
  8. Why do I see additional DNA bands on my gel after a restriction digest?
  9. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?

Protocols

  1. Optimizing Restriction Endonuclease Reactions
  2. Double Digest Protocol with Standard Restriction Enzymes
  3. Time-Saver Protocol for Restriction Enzyme Digests

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.
  • 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.