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S U P P O R T
 

Protocols for RNA Electrophoresis

Note
RNA ladders, as well as any RNA, are extremely sensitive to degradation by ribonucleases. The use of fresh electrophoresis buffers, freshly poured gels, diethyl pyrocarbonate-treated solutions and protective gloves is recommended. Gloves are also necessary when solutions containing ethidium bromide are handled.

Preparation of RNA ladder for electrophoresis

For RiboRuler™ RNA ladders

  1. Mix 1 volume of RNA ladder and 1 volume of the 2X RNA Loading Dye.
  2. Heat at 70°C for 10 min.
  3. Chill on ice and spin down prior to loading on a gel.
  4. Load 0.5 µl of the prepared RNA ladder for every mm of gel lane width (4 µl/8 mm).

For ready-to-use RiboRuler™ RNA ladders

  1. Heat RNA ladders at 70°C for 10 min.
  2. Chill on ice for 3 minutes and spin down prior to loading on a gel.
  3. Load 0.5 µl of the RNA ladder for every mm of gel lane width (4 µl/8 mm).

Note
RNA ladders prepared as described above are not suitable for glyoxal/DMSO agarose gel electrophoresis. To prepare RNA ladders for glyoxal/DMSO agarose gels, please refer to the recommendations for glyoxal/DMSO agarose electrophoresis.

Preparation of RNA sample for electrophoresis
  1. Mix 1 volume of the 2X RNA Loading Dye and 1 volume of RNA sample.
  2. Heat at 70°C for 10 min.
  3. Chill on ice for 3 minutes and load onto the gel.

Note
RNA samples prepared as described above are not suitable for glyoxal/DMSO agarose gel electrophoresis. To prepare RNA samples for glyoxal/DMSO agarose gels, please refer to the recommendations for glyoxal/DMSO agarose electrophoresis.

Non-denaturing agarose gel electrophoresis
  1. Dilute 50X TAE buffer or 10X TBE buffer to a 1X concentration immediately before use.
    Note
    Use TBE buffer for analysis RNA bands smaller than 1500b. For larger RNA, use TAE buffer.
  2. Prepare 1% TopVision™ LE GQ Agarose gel in 1X TAE or TBE according to the recommended protocol.
    Note
    For optimal results, add ethidium bromide to both the agarose gel and electrophoresis buffer at a final concentration of 0.5 µg/ml.
    Alternatively, the gel can be stained after electrophoresis by immersing it into a 0.5 µg/ml ethidium bromide solution for 20 min, or by any other RNA staining technique.
  3. Place the gel into an electrophoresis apparatus containing the appropriate buffer.
  4. Heat the RNA samples and ladder at 70°C for 10 min, and then chill on ice for 3 minutes. Load onto the gel.
  5. Run electrophoresis at 5 V/cm until the bromophenol blue runs approximately two-thirds of the way down the gel.

Denaturing formaldehyde gels in MOPS buffer
  1. Freshly prepare 10X MOPS buffer:
    0.4 M MOPS (pH 7.0),
    0.1 M Sodium acetate,
    0.01 M EDTA (pH 8.0).
  2. Prepare 1% TopVision™ LE GQ Agarose gel as follows:
    • stir 1g of agarose powder in 72 ml of deionized water
    • melt the agarose, and then add 10 ml of 10X MOPS buffer and mix
    • when the agarose solution cools to 60°C, add 18 ml of fresh formaldehyde (37%) in a fume hood and mix thoroughly
    • pour the gel.
  3. Place the gel into an electrophoresis apparatus containing 1X MOPS buffer.
  4. Heat the RNA samples and ladder at 70°C for 10 min, and then chill on ice for 3 minutes. Load onto the gel.

Note
There is no need to stain the gel as ethidium bromide present in 2X RNA Loading Dye is sufficient for visualization under UV light.

Denaturing glyoxal/DMSO gels in sodium phosphate buffer
  1. Prepare thick 1.0% TopVision™ LE GQ Agarose gel in 0.01 M sodium phosphate buffer, pH 7.0.
  2. Place the gel into an electrophoresis apparatus containing 0.01 M sodium phosphate buffer, pH 7.0.
  3. Prepare for loading 25 µl aliquots of the ladder/samples by adding:
    Glyoxal (40% solution) 4.5 µl
    DMSO 12.5 µl
    0.1 M sodium phosphate buffer, pH 7.0 2.5 µl
  4. Mix and add:
    RiboRuler™ RNA Ladder 3 µl
    2X RNA Loading Dye 1 µl
    DEPC-treated Water to 25 µl
  5. Incubate for 1 hour at 50°C and then cool down to room temperature.
  6. Load the samples on a gel.
  7. Run electrophoresis at 5 V/cm until the bromophenol blue runs approximately two-thirds of the way down the gel
  8. Stain the gel in ethidium bromide solution (final concentration 0.5 µg/ml) in 0.5 M ammonium acetate for 15-30 min
  9. Wash the gel in fresh 0.5 M ammonium acetate solution for 15-30 min.

Denaturing polyacrylamide/urea gels in TBE buffer
  1. Prepare 20 ml of a 5% polyacrylamide gel containing 7 M urea by adding:
    47.5% acrylamide: 2.5% bis-acrylamide solution 2 ml
    10 M urea 14 ml
    10X TBE buffer 2 ml
    10% freshly prepared ammonium persulfate 0.2 ml
    deionized water 1.8 ml
  2. Mix and add 10 µl TEMED
  3. Mix again and pour the gel carefully avoiding the formation of air bubbles.
  4. Insert the comb into the acrylamide and allow the gel to polymerize for at least 1 hour.
  5. Fill the electrophoresis apparatus with 1X TBE buffer
  6. Heat the RNA samples (premixed with equal volume of 2X RNA Loading Dye) and ladder at 70°C for 10 min, and chill on ice for 3 minutes. Load onto the gel.
  7. Run electrophoresis at 8 V/cm for about 1 hour.
  8. Soak the gel for about 15 minutes in 1X TBE to remove urea prior to staining.
  9. Stain the gel in 0.5 µg/ml ethidium bromide in 1X TBE solution for 15 min.
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Updated vasario 01, 2008 10:42