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.
    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.
    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.
    What stripping protocol should I use with Immobilon-Ny+?A variety of stripping protocols work with Immobilon-Ny+. DNA probes can be stripped form the membrane by incubation of the blot in 0.4 N NaOH at 45° for 30 min. followed by neutralization for 15 minutes at room temperature in 0.1X SSC, 0.1% (w/v) SDS, 0.2 M Tris-HCl, pH 7.5. Alternatively, the probe can be stripped by placing the blot in boiling 0.5% (w/v) SDS and allowing the solution to cool to room temperature.
    How do I strip the Immobilon Ny+ when doing Northerns with RNA probes?Heat 0.1% SDS to boiling, remove from heat. Place blot in solution and agitate gently for 15 minutes. Repeat this once. Seal the blot in a plastic bag. DO NOT use NaOH - signal intensity increases dramatically upon reprobing if 50mM NaOH is used.
    Can I dry the Immobilon NY+ membrane?The Immobilon NY+ membrane should not be dried during the hybridization, washing or film exposure steps if the membrane will be reprobed. Drying causes the probe to irreversibly bind to the membrane.
    For colony and plaque lifts, is it necessary to wet and autoclave the membrane prior to the lift?It is only necessary if you are worried about carrying over microbes that could possibly be present and alive on the membrane. If you choose not to autoclave the membrane, then the membrane can be placed dry onto the plate for the lift directly. The plates should be chilled first at 4 degrees C for about 30 minutes prior to the lift in order to prevent the medium from lifting up with the membrane.
    Is there a difference in the binding mechanism for the Immobilon-Ny uncharged nylon membrane compared to the Immobilon- Ny+ charged nylon membrane?Yes, the charged Immobilon-Ny+ binds primarily by ionic interactions and by covalent interactions only after UV fixation. The uncharged Immobilon-Ny binds primarily by electrostatic interaction and covalent after UV fixation.

    User Guides

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