• 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

        0.1-2 Kb RNA Ladder

        互联网

        2779

        实验步骤

         

        1. Formaldehyde Denaturing Gels

            1) Pre-run a horizontal 1.5-2.5% agarose gel (2-3 mm, lane width) containing 6% (w/v) formaldehyde, 20 mM MOPS (pH 7.0), 1 mM EDTA submerged in buffer (20 mM MOPS, pH 7.0; 1 mM EDTA) for 10 minutes at 100 V.

            2) To 3 μg (3 μl) of ladder, add loading buffer (recommended final loading buffer concentration is 20 mM MOPS; pH 7.0, 6% w/v formaldehyde, 31% v/v deionized formamide, 1 mM EDTA, 0.0125% xylene cyanol and 0.0125% bromophenol blue) and mix well.

            3) Denature the ladder in loading buffer at 70°C for 10 minutes. Keep on ice for 1-2 minutes to quench.

            4) Load ladder and your RNA samples on the pre-run gel from Step 1.

            5) Perform electrophoresis at 100 V until the bromophenol blue dye has migrated two-thirds the gel length.

            6) Stain the RNA ladder with:

                  i. 0.5 μg/ml EtBr solution for 10 minutes and detain the gel in deionized water for 10 minutes OR

                 ii. 1:10000 dilution of SYBR® Green II RNA Gel Stain (cat. no. S7564) in TBE for 20-40 minutes. No destaining is required.

            7) Visualize ladder bands on a UV transilluminator (302 nm) and photograph using a gel documentation system. The RNA bands may fade and disappear on prolonged exposure to UV light.

        2. Glyoxal Gels

            1) Denature 3 μg (3 μl) RNA Ladder in loading buffer (recommended final loading buffer concentration is 1 M deionized glyoxal, 50% (v/v) DMSO, 10 mM sodium phosphate (pH 7.0), 0.01% xylene cyanol, 0.01% bromophenol blue for 30 minutes at 50°C. Keep on ice for 1-2 minutes to quench.

            2) Load your samples on a horizontal 2.5% agarose gel containing 10 mM sodium phosphate (pH 7.0), 2 mg/ml sodium iodoacetate. Run the gel submerged in 10 mM sodium phosphate (pH 7.0) at 160 V for 2 h.

            3) Stain with EtBr or SYBR® Green II, visualize, and image gel.

        3. Acrylamide Gels

        1) Denature 0.5 μg (5 μl of 1:10 dilution) RNA Ladder in 2X Novex® TBEUreaSample Buffer (cat. no. LC6876) at 70°C for 5 minutes. Keep on ice for 1-2 minutes to quench.

        2) Load ladder and your RNA samples onto a 1 mm thick Novex® 6% TBE-Urea mini-gel (cat. no. EC6865BOX) Run the gel at 180 V for 1-2 h.

        3) Stain with EtBr or SYBR® Green II, visualize, and image gel.

        4. cDNA Synthesis

        A protocol for cDNA synthesis using 0.1-2 Kb RNA Ladder is described below. Other protocols are suitable.

        1) Incubate 2 μg (2 μl) RNA ladder with 5 μg anchored Oligo(dT)20 primer at 70°C for 5 minutes. Keep on ice.

        2) Combine the RNA-oligo dT complex from Step 1 with 1X 1st strand cDNA synthesis buffer supplemented with 10 mM DTT, 500 μM dNTP, 2 U RNaseOUT™ (cat. no.10777-019), 1-3 μCi -P32-dCTP (3000Ci/mmole) or Chromatide®Alexa Fluor® 546-14-UTP (cat. no. C-11404) in a reaction volume of 18 μl.

        3) Add 400 U of SuperScript™ III Reverse Transcriptase (cat. no.18080-093). Adjust final reaction volume to 20 μl. Incubate at 50°C for 1 hour.

        4) Add 10 μl 0.5 M EDTA, pH 8.0 to terminate the reaction and then add 20 μl STE buffer (150 mM NaCl, 10 mM Tris-Cl, pH 8.0, 1 mM EDTA).

        5) Remove unincorporated NTPs by gel filtration with G-50 columns.

        6) Mix a fraction of the purified cDNA reaction with 10X alkaline agarose loading dye (300 mM NaOH, 2 mM EDTA, 20% glycerol, 10% saturated thymol blue).

        7) Analyze samples on a 3% alkaline agarose gel containing 30 mM NaOH, 2 mM EDTA pH 7.5 and electrophoresed using 30 mM NaOH, 2 mM EDTA pH 7.5.

        8) Dry the gel in a heated vacuum drier for 1.5 hours and expose the gel to a film or a phosphoimager plate.

        注意事项

         

        1. To avoid repeated freezing and thawing, aliquot the ladder in small volumes and store at -80°C

        2. Always wear gloves, use sterile, RNase-free tips, tubes and plasticware, and use aseptic techniques

        ad image
        ad image

        相关产品推荐

        T7 RNA polymerase 多克隆抗体 29943-1-AP

        T7 RNA polymerase 多克隆抗体 29943-1-AP

        ￥1350

        MCM-41分子筛，孔径2.5nm，粒径1-2微米，比表面积(m2/g)：600-800 全硅，阿拉丁

        MCM-41分子筛，孔径2.5nm，粒径1-2微米，比表面积(m2/g)：600-800 全硅，阿拉丁

        ￥601.90

        100 bp DNA分子量标准(100bp DNA Ladder)

        100 bp DNA分子量标准(100bp DNA Ladder)

        ￥180

        1 kb DNA分子量标准

        1 kb DNA分子量标准

        ￥100

        LRRFIP1/LRRFIP1蛋白Recombinant Human Leucine-rich repeat flightless-interacting protein 1 (LRRFIP1)重组蛋白GC-binding factor 2TAR RNA-interacting protein蛋白

        LRRFIP1/LRRFIP1蛋白Recombinant Human Leucine-rich repeat flightless-interacting protein 1 (LRRFIP1)重组蛋白GC-binding factor 2TAR RNA-interacting protein蛋白

        ￥1344

        相关问答

        问

        1-2mmol/L的薄片是什么意思呢

        4 回答 470 围观
        2023-07-18

        问

        【求助】DNA ladder

        3 回答 367 围观
        2022-11-24

        问

        DNA ladder能当marker使用吗

        3 回答 1329 围观
        2022-05-24

        相关方法

        DNA ladder法

        DNA ladder法检测细胞凋亡

        RNA 提取

        推荐阅读

        Kb kilobase

        【求助】关于NF_KB的一点疑惑？？？

        制作DNA ladder常见问题

        无忧采购 轻松科研

        无忧采购 轻松科研

        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