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Transferencias "Northern" y "Southern", y de colonias/placas de lisis

A choice of transfer membranes for nucleic acid applications.

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Membranas de transferencia Immobilon-NC libre de surfactante Borrar Clasificación y Filtrado
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HATF0825082 mm 50 Precios y disponibilidad

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Membranas de transferencia Immobilon-Ny+ – Hojas y rollos Borrar Clasificación y Filtrado
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INYC0001030 cm x 3.3 m 1 Precios y disponibilidad

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Membranas de transferencia Immobilon-NC – Hojas y rollos Borrar Clasificación y Filtrado
Número de referenciaicon Dimensiones del filtroicon Tamaño de envaseicon
HATF0001033 cm x 3 m 1 Precios y disponibilidad

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    Documentation

    Licencias necesarias e Información técnica

    Cargo
    Optimization of northern blotting on Immobilon-Ny+

    Ficha técnica

    Cargo
    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

    Referencias bibliográficas

    Visión general referencias
    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

    Preguntas frecuentes

    PreguntaRespuesta
    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 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.
    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.
    Can Immobilon-Ny be autoclaved before colony or plaque lifts? Yes. Sterilize the membrane as follows: Wet the membrane by laying it on top of Milli-Q water and letting the water slowly absorb through the membrane (this will prevent airlock, which could occur if the membrane were quickly immersed in the water). Sandwich the wet membranes between dry sheets of 3MM filter paper. Wrap the pile of membranes in aluminum foil. Autoclave at 15 lb./sq. in (1.05 kg/sq. cm) at 121 degrees C on a liquid cycle (slow exhaust).
    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 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 many times can I strip and reprobe Immobilon-Ny+? Signal has been clearly observed after 13 rounds of reprobing.
    I would like to do a northern blot. Which membrane should I use and what conditions does Millipore recommend? RNA blotting as well as plaque lifts can be done on both nitrocellulose and nylon membranes. We recommend the Immobilon NY+ membrane for all nucleic acid blotting protocols. In particular, we recommend the Northern blotting protocol outlines in the book Molecular Cloning, Cold Spring Harbor Labs.
    What is the optimal UV energy for a maximum hybridization signal on the Immobilon NY membrane? Optimal UV cross-linking energy varies depending on the size of the colony. To optimize the UV energy for maximum hybridization signal use 5,000 uJoules/cm2 at 254nm for direct spotting of bacterial suspension on a membrane surface without further culture. 60,000 uJoules/cm2 at 254nm for <1-2mm diameter colonies. 120,000 uJoules/cm2 at 254nm for 3-5 mm diameter colonies.
    Why should I use the Immobilon-Ny uncharged blotting membrane instead of the Immobilon-Ny+ charged blotting membrane? The uncharged nylon blotting membrane has reduced background issues with chemifluorescence and northerns than the charged nylon membrane, the uncharged nylon membrane is less expensive, and the uncharged nylon membrane is ready to use with current protocols for faster transfer, immobilization and hybridization applications without optimization requirements.
    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.
    What can I do to reduce high background on my Southern or northern blot when using Immobilon-Ny+ or Immobilon-Ny? To minimize background, use >18.2 megaohm water when making solutions. Filter solutions before use with a 0.22 or 0.45 um filter (Stericup filter unit with 0.22 um Millipore Express membrane). A modified Church hybridization solution (0.5 M sodium phosphate, pH 7.1, 2 mM EDTA, 7% (w/v) SDS, 0.1% (w/v) sodium pyrophosphate) is recommended over Denhardt's solution (33 - 50% formamide may be added when performing northerns to prevent cross-hybridization of an RNA probe to ribosomal RNA). If working in high humidity climate, it may be necessary to ensure complete dryness of membrane by baking blot in vacuum oven prior to UV fixation.
    How do I choose between Immobilon-Ny+ and Immobilon-Ny when performing Southern or northern blots or plaque/colony lifts? If you are looking for a membrane that is ready to use in an existing protocol without the need to optimize, is economical and exhibits low background, Immobilon-Ny is recommended. It is ideal when looking for qualitative results, like the presence of absence of a band. If you are looking for maximum sensitivity, especially when looking for quantitative results (measuring signal intensities), Immobilon-Ny+ is recommended. There are optimized protocols available to obtain best results.
    What is the thickness of Immobilon-Ny+? The membrane is 6 mils to 7.5 mils (152 - 190.5 microns) minimum to maximum thickness.
    If I want to optimize/change the settings for UV fixation of DNA or RNA on Immobilon-Ny+, how to I set my Stratalinker? To set the Stratalinker: Turn the power on. Make sure the window shows "0000". If not, hit the "Reset" button. Press the "Energy" button. Make sure the "X 100" light is on. Enter desired UV setting (i.e. "50" for 5,000 uJoules/cm2). See TN054, TN071 for DNA fixation settings; TN110 for RNA settings. Hit "Start" button. UV cross-linking is complete within a few seconds, then Stratalinker will beep. Press "Reset" button to stop the beeping.
    How long can I store my northern blot of Immobilon-Ny+ after cross linking? The duration for storage depends on the quality of the blot (free from RNase), the sensitivity required (abundance of target RNA and how much sample RNA was loaded), etc. Depending on these conditions, the blots may be stable at 4 degrees C for a month with the band of interest still able to be detected without any signal decrease. Place the dried northern blots between clean 3MM filter papers in a clean plastic bag and store them at 4 degrees C in the dark. Alternatively, the dried blots may be stored at -20 degrees C. Wet blots are not recommended for long term storage.
    What type of sodium phosphate is used to make Modified Church's Buffer for Northern blotting hybridization? You need to use both monobasic and dibasic sodium phosphate together to make a 0.5M solution adjusted to pH 7.1. For example, to make 1 liter of Modified Church's Buffer, mix:
    • 32.9 g Sodium Phosphate- monobasic (MW 137.99)
    • 70.1 g Sodium Phosphate- dibasic (MW 268.07)
    • 70.0 g SDS

    -
    1.0 g Sodium pyrophosphate

    - 4.0 ml 0.5M EDTA


    Fill to 1 L with Milli-Q water

    Manuales del usuario

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