Novex™ TBE-Urea Gels, 10%

Here are some suggestions for your experiments:

- The RNA samples may have been degraded by RNases. Use standard precautions to prevent contamination.
- Make sure samples are heated for 3 minutes at 70°C just prior to loading. If this is not done, the oligonucleotides will not be fully denatured, which may result in a smeared background.
- Be sure to vigorously flush urea out of sample wells just prior to loading the sample. Urea will continually seep from the gel into the well. Urea is very dense and will force the sample into a ball.
- A sample volume over 10 µL may result in smearing. This volume is less than may be used with agarose gels.
- If ultrapure water (18 milliohms) is not used, smeared bands and high background may result.
- Check to make sure the running and sample buffers have been prepared and diluted correctly.
- Make sure to run the bromophenol blue until it reaches the slot. Stopping the run short can result in less than optimal results.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

There are many sample buffer formulations used; however, we have found a distinct difference in the band appearance depending on the sample buffer composition. After evaluating urea, formamide, and various buffer systems, we found that the sharpest, flattest bands were obtained with a urea, Ficoll, and TBE buffer solution. Sample buffers made with formamide provided fuzzy, indistinct bands.

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Yes, for ethidium bromide staining, soak the gel in a 2 µg/mL solution of ethidium bromide in ultrapure water for 20 minutes. Destain by rinsing with three successive 10-minute rinses of ultrapure water. Visualize bands under UV light

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To help you choose the TBE polyacrylamide gel that will provide optimal separation results for your nucleic acid fragment, we have designed gel migration charts for each gel type offered. Please check the TBE gel migration charts on page 74 of the Novex Pre-Cast Gel Electrophoresis Guide (https://tools.thermofisher.com/content/sfs/manuals/electrophoresisguide_man.pdf).

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Here is the protocol you can follow:
(1) Run the gel. Locate the DNA of interest by autoradiography or by examination of ethidium bromide stained gels.
(2) Use a razor blade to cut out the upper 1/2 to 2/3 of the band of interest.
To recover a fragment of DNA identified by autoradiography, cut out from the X-ray film a small rectangle encompassing the image of the fragment. Align the film over the gel and cut out the segment of polyacrylamide bordered the rectangular hole in the film.
(3) Transfer the gel slice to a microcentrifuge tube. Use a sterile glass rod to crush the slice against the wall of the tube.
(4) Calculate the volume of the slice and add 1 to 2 volumes of elution buffer to the microcentrifuge tube.
Elution buffer: 0.5 M ammonium acetate, 10 mM magnesium sulfate, 1 mM EDTA (pH 8.0), 0.1% SDS
It is convenient if the volume of elution buffer is no greater than 0.5 mL since the eluted fragment of DNA can then be precipitated with ethanol in a single tube.
(5) Close the tube and incubate at 37 degrees C on a rotary platform. Small fragments of DNA (500 bases) are eluted in 3 to 4 hours.
(6) Centrifuge the sample at 12,000 g for 1 minute at 4 degrees C in a microcentrifuge. Transfer the supernatant to a fresh microcentrifuge tube, being careful to avoid transferring fragments of polyacrylamide.
(7) Add additional 0.5 mL volume of elution buffer to the pellet of polyacrylamide, vortex briefly, and re-centrifuge. Combine the two supernatants.
(8) Remove any remaining fragments of polyacrylamide by passing the supernatant through a disposable plastic column or a syringe barrel containing Whatman GF/C filter or packed siliconized glass wool.
(9) Add 2 volumes of ethanol at 4 degrees C and store the solution on the ice for 30 min. Recover the DNA by centrifugation at 12,000 g for 10 min at 4 degrees C in a microcentrifuge.
(10) Re-dissolve the DNA in 200 µL of Tris-EDTA (TE) buffer (pH 7.6), add 25 µL of 3M sodium acetate (pH 5.2) and re-precipitate the DNA with 2 volumes of ethanol as described in step 9. TE Buffer (pH 7.6): 10 mM Tris HCl (pH 7.6), 1mM EDTA (pH 8.0).
(11) Carefully rinse the pellet once with 70% ethanol and redissolve the DNA in TE buffer (pH 7.6) to a final volume of 10 µL.
(12) Check the amount and the quality of the fragment by polyacrylamide gel electrophoresis.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.