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    0.1-2 Kb RNA Ladder

    互联网

    2548

    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

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