Northern and Southern Blotting, and Colony and Plaque Lifts

A choice of transfer membranes for nucleic acid applications.

Overview

Specifications

Ordering Information

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Immobilon-NC Surfactant-free Transfer Membranes – Filter Discs Clear Sorting & Filtering
Catalogue Numbericon Filter Diameter (⌀)icon Pack Sizeicon
HATF0825082 mm 50 Show Pricing & Availability
HATF0855085 mm 50 Show Pricing & Availability

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Immobilon-Ny+ Transfer Membranes – Sheets and Roll Clear Sorting & Filtering
Catalogue Numbericon Filter Dimensionsicon Pack Sizeicon
INYC0001030 cm x 3.3 m 1 Show Pricing & Availability

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Immobilon-NC Transfer Membranes – Sheets and Roll Clear Sorting & Filtering
Catalogue Numbericon Filter Dimensionsicon Pack Sizeicon
HATF0001033 cm x 3 m 1 Show Pricing & Availability

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    Documentation

    Technical Info

    Title
    Optimization of northern blotting on Immobilon-Ny+

    Data Sheet

    Title
    Immobilion-Ny+ Blotting Membrane: Charged nylon blotting membrane for superior results from nucleic acid protocols
    Immobilon Transfer Membranes: For superior protein and nucleic acid blots

    References

    Reference overview
    ImmobilonTM-Ny+ nucleic acid blotting membrane: An advanced nylon membrane optimized for superior fixation and reprobing
    Mansfield Michael A(a); Mabuchi Masaharu; MacDonald Constance G; Pluskal Malcolm G
    Biotechniques v 27 pg 1253-1257 Dec., 1999  1999

    Comparison of antigen binding capabilities of various membrane filters and filter papers in dot immunoassay
    Lin Maw-Yeong(a); Tung Mon-Chu; Sung Haw-Tsung
    Chinese Journal of Microbiology and Immunology (Taipei) v29 pg 108-115 1996  1996

    FAQ

    QuestionAnswer
    What is the Immobilon NY Blotting Membrane?Immobilon NY membrane is an uncharged 0.45um modified nylon blotting membrane. The membrane was developed specifically for nucleic acid blotting applications, such as Southern and Northern blotting, colony and plaque lifts.
    How sensitive is the DNA detection on Immobilon-Ny+?Lambda DNA has been detected down to 0.42 picograms.
    What is Immobilon-Ny+?Immobilon-Ny+ is a web-supported, positively charged, hydrophilic nylon blotting membrane. The rated pore size of the membrane is 0.45um.
    I am using a kit from NE Biolabs kit which recommends the Immobilon S. Since this membrane has been obsoleted, which membrane shall I use?We have worked with New England Biolabs, and they have evaluated our Immobilon NY+ with their kit. They had excellent results and recommend the Immobilon NY+ as a replacement for Immobilon S.
    What are the conditions I should use for UV cross-linking DNA to Immobilon-Ny+?The following guidelines should be observed for successful UV cross-linking to Immobilon-Ny+:
    • The DNA should be denatured before being filtered or transferred onto Immobilon-Ny+.
    • The membrane should be dried completely before cross-linking.
    • UV cross-link filtered DNA at 1700-5000 microJoules/cm2, and blotted DNA at 5000 microJoules/cm2. The preferred wavelength is 254 nm.
    Does it matter what type of water I make up the reagents for Northern and southern blots?Yes, use Milli-Q water or equivalent water with >18.2 megaohm resistivity should be used to prepare all reagents. Water systems should be well maintained to avoid the build-up of organic contaminants, i.e., pyrogens.

    Some facts to keep in mind:
    • Nucleases will digest probes.
    • Particulates can lead to high backgrounds by serving as sites for non-specific binding of probe molecules.
    • Heavy metal ions can bind to DNA and RNA, altering their structure and inhibiting hybridization.
    • Pyrogens will inhibit horseradish peroxidase, which is used as the enzyme in many non-radioactive kits.
    • Variable water quality can cause variable results that are independent of the membrane.
    What are the advantages of using nylon for performing Southern and Northern blots?DNA and RNA can be permanently bound to nylon, as opposed to nitrocellulose where the DNA may wash off during hybridization and post-hybridization washes. Nylon is also more durable than nitrocellulose, which makes it well suited for numerous rounds of stripping and reprobing.
    How do I fix the DNA on Immobilon-Ny+?For best results, Millipore recommends UV cross-linking at 5000 microJoules/cm2 for blotted DNA and 1700-5000 microJoules/cm2 for filtered DNA. UV energies in excess of the recommended levels will cause a rapid reduction in hybridization signal. The membrane should be dried prior to UV cross-linking.
    What transfer method should I use with Immobilon-Ny+?Maximum sensitivity is achieved with capillary transfer in 20X SSC buffer followed by fixation of the DNA by UV cross-linking. While alkaline transfer is compatible with Immobilon-Ny+, the hybridization efficiency is reduced. UV cross-linking has no effect on hybridization of DNA transferred under alkaline conditions.
    How many times can I strip and reprobe Immobilon-Ny+?Signal has been clearly observed after 13 rounds of reprobing.

    User Guides

    Title
    Immobilon-NY Transfer Membrane User Guide
    Immobilon-Ny+ Transfer Membrane