• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        NorthernBlot--northern杂交

        互联网

        3121

        Northern Blot

        Preparation of Formaldehyde Agarose Gel

        The gel conditions (1% agarose, 1X MOPS, 6.3% formaldehyde) are designed for ~4 hours of electrophoresis. If longer times are necessary, the formaldehyde concentration should be increased.

        1.Heat agarose in water to get the agarose into solution.

        2.Cool the agarose to approximately 55o C.

        3.Add the appropriate volume of 10X MOPS and formaldehyde for final concentrations of 1X and 6.3%, respectively.

        4.Pour the gel in the hood to avoid toxic formaldehyde fumes.

        Preparation of RNA Samples

        Generally, 10-30 μg of RNA is loaded per lane.

        1.Add the appropriate volume of RNA to an eppendorf tube.

        2.Dry down the samples in a Speed Vac.

        3.Resuspend the samples in 25 μl of sample buffer and heat at 65oC for 10 minutes.

        4.Add 2 μl of the dye solution and load gel.

        Running Conditions

        Run at 3.5 volts/cm for 3-4 hours, with recirculating buffer (1X MOPS). It is also acceptable to place the gel apparatus on two stirring blocks to recirculate the buffer. However, under these conditions it may be necessary to weigh down the gel with cheesecloth to prevent the gel from floating off the gel running platform.

        Transfer of RNA to Membrane

        1.After electrophoresis is complete, wash the gel twice in a large volume of deionized water to remove the formaldehyde.

        2.Transfer the RNA by capillary blotting to nitrocellulose using a 20X SSC reservoir. Do not rinse the membrane after transfer.

        3.Cross link RNA to nitrocellulose using the Stratalinker.

        Hybridization Conditions

        1.Use 100 μl of hybridization solution per square cm of membrane.

        2.Prehybridize at 42℃for 1-2 hours in hybridization solution (see below) containing freshly boiled salmon sperm DNA at a final concentration of 100 ug/ml, and 1 ml of 50% dextran sulfate per every 5 ml of hybridization solution.

        3.Add boiled probe to the prehybridization and continue 42℃incubation overnight.

        Washing Conditions

        Usually, two washes in 2X SSC, 0.1% SDS for 10 minutes each at room temperature is sufficient. However, if necessary, washes in 0.1 X SSC, 0.1% SDS 10 minutes at room temperature or 42℃may be done to reduce background hybridization.

        Solutions

        10X MOPS Buffer (store at 4oC in the dark)

        0.2 M MOPS, pH 7

        50 mM Sodium acetate

        10 mM EDTA

        Sample Buffer

        50% formamide

        2.2 M formaldehyde

        1X MOPS

        10X Running Dye Solution

        50% glycerol

        0.3% Bromphenol Blue

        0.5 ug/ul Ethidium bromide

        Salmon Sperm DNA

        10mg/ml in water

        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序