• 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

        DNA Sequencing Gels

        互联网

        929

         

        DNA Sequencing Gels

        Buffers and gel solutions

        Long Ranger: we started using this in early 1995. Great stuff; the best thing is that the gels are not sticky after drying, even without removal of the urea. Long Ranger Gel Solution is available from many scientific supply companies and is a JT Baker reagent, cat # 4730-02 (50% concentrate).

         

        The table below is used by our lab, and is sufficient for a standard 35 x 43 cm x 0.4 mm gel (we use the Owl boxes). The Long Ranger solution comes with a troubleshooting guide; a few of the electrophoresis parameters that have been demonstrated important for us are listed below.

        Long Ranger Gels
          5% in 1X TBE 5% in 1.2X TBE 6% in 1X TBE
        urea 40.4 g 40.4 g 40.4 g
        TBE 13.3 ml 6X
        (8 ml 10X)
        16 ml 6X
        (9.6 ml 10X)
        13.3 ml 6X
        (8 ml 10X)
        Long Ranger (50%) 8 ml 8 ml 9.6 ml
        water 30.5 ml
        (35.8 ml)
        27.8 ml
        (34.2 ml)
        28.9 ml
        (34.2 ml)
        ammonium persulfate 70 mg 70 mg 70 mg

         

        1. Mix to dissolve urea, filter through Whatman 3MM filter or equivalent.
        2. Add 25 ul TEMED, and pour gel.
        Electrophoresis
        1. For maximum base reads, use 1.2X TBE in the gel and 0.6X TBE as the electrophoresis buffer.
        2. Run gels at 55 W. Wattage must not exceed 55-60 W even with large 40 x 40 cm plates or a temperature of 50 C.
        3. A 10 min prerun is sufficient
        4. Maintain gel temperature between 40-50 C.
        5. Do not heat samples excessively during denaturation step. 2 min at 75 C is enough, and it is not necessary to heat again prior to a second or third loading.
        6. We have had the best results with 4.5 and 2 hour runs, at 55 W.
        Electrophoresis Buffers (TBE)
          10X (per liter) 6X (per liter)
        Tris base 108 g 64.8 g
        Boric acid 55 g 33 g
        disodium EDTA 9.3 g 5.58
        (The TBE should be fresh-if the boric acid precipitates, make a fresh stock--we find that a 6x solution lasts longer before precipitating)

        Older (pre-Long Ranger Gels) Method

        10X EB
        164.0 g Tris-OH
        27.5 g Boric Acid
        7.45 g disodium EDTA
        water to 1 l

        Acrylamide stock

        38% Acrylamide
        2% bis-acrylamide

        Gels (volume is for standard size sequencing gels)

         

         

        8% gel:
        40.4 g urea
        27.0 ml water
        16.8 ml 38/2 acrylamide
        8.0 ml EB
        6% gel:
        40.4 g urea
        31.2 ml water
        12.6 ml 38/2 acrylamide
        8.0 ml EB
        5% gel:
        40.4 g urea
        33.5 ml water
        10.5 ml 38/2 acrylamide
        8.0 ml EB

        Dissolve by heating slightly, degas if necessary. Add 0.7 ml 10% ammonium persulfate, and 25 ul TEMED, and pour gel immediately.

         

        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