Novagen RNA 标记剂产品说明书

Novagen
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RNA Gel Electrophoresis
About the Kits
Perfect RNA™ Markers, 0.1–1 kb
25 lanes 69924-3 Perfect RNA Markers, 0.2–10 kb
25 lanes 69946-3 RNA Markers, 0.36–9.5 kb
25 lanes
69211-3 RNA Sample Buffer 1 ml 70606-3
Description
An RNA preparation can be rapidly assessed for quality and size by gel electrophoresis, followed by
staining with ethidium bromide or SYBR ® Green II and comparison to size standards. The Perfect RNA
Markers™ are a mixture of defined RNA molecules suitable as standards for gel analysis of RNA under
native or denaturing conditions. The markers are of easy-to-remember sizes and cover convenient size
ranges. Perfect RNA™ Markers, 0.1–1 kb, contain seven defined RNA molecules with lengths of 100, 200,
300, 400, 600, 800, 1000 nucleotides. Perfect RNA Markers, 0.2–10 kb, contain nine defined RNA
transcripts: 200, 500, 1000, 1500, 2000, 3000, 4000, 6000, 10,000 nucleotides. The original Novagen RNA
Markers, 0.36–9.5 kb, contain eight equal-intensity RNA bands: 363, 562, 872, 1898, 2800, 3911, 6225,
9488 nucleotides. This popular mixture is useful for the same applications as Perfect RNA Markers, 0.2–10
kb. Each vial contains 50 µl of RNA Markers, enough for 25 gel lanes.
RNA Sample Buffer is a convenient, premixed, formamide-containing sample buffer for experimental
samples and Perfect RNA Markers. This sample buffer is suitable for native agarose gels and denaturing
urea-containing polyacrylamide gels.
© 2006 EMD Biosciences, Inc., an affiliate of Merck KGaA, Darmstadt, Germany. All rights reserved. The Novagen ® name is a registered trademark of EMD Biosciences, Inc in the United States and in certain other jurisdictions. Perfect RNA™ is a trademark of EMD Biosciences, Inc. SYBR ® is a registered trademark of Molecular Probes.
Components
Perfect RNA™ Markers, 0.1-1kb
• 50 µl Perfect RNA™ Markers, 0.1-1kb
Perfect RNA Markers, 0.2-10kb
• 50 µl Perfect RNA Markers, 0.2-10kb
RNA Markers, 0.36-9.5kb
• 50 µl RNA Markers, 0.36-9.5kb
RNA Sample Buffer
• 50 µl RNA Sample Buffer
Storage
Store components at -70°C.
RNA Gel Electrophoresis
Native gels (agarose in 1X TBE) are easy to run and RNA is stained and photographed with higher
sensitivity than in denaturing gels. Although samples are denatured prior to loading, effects of RNA
secondary structure formed during electrophoresis can lead to somewhat anomalous migration. Denaturing
gels (formaldehyde-agarose or polyacrylamide-urea) reduce effects of RNA secondary structure and provide
more accurate size determinations than native gels, but are difficult to run and stain. Denaturing gels are
recommended for Northern blots as RNA binding to membrane supports is more efficient than from native
gels. For staining before transfer, SYBR Green II is preferred to ethidium bromide.
Avoiding Ribonuclease Contamination
Ribonucleases present in the environment, and within the RNA sample, can rapidly degrade RNA, resulting
in poor quality. To avoid RNA degradation, several precautions should be observed:
• All materials coming in contact with the sample must be sterile and RNase-free. Use sterile disposable
pipets, pipet tips, and tubes. Use sterile technique at all times.
• Wear gloves during entire procedure to avoid introducing RNase contaminants to the samples.
• Use RNAse-free water. Treat water by adding DEPC to Milli-Q, or equivilant, deionized water, to a final
concentration of 0.1% (0.001 vol), mixing well, and heating to 70°C for 1 h. Observe proper safety
precautions, including use of a fume hood. DEPC is a powerful acylating agent and forms ethyl
carbamate, a potent carcinogen, when exposed to ammonia.
• Reserve reagents exclusively for RNA work. Avoid using any reagents that may have been used for
other work. If possible, separate any laboratory procedures that require the use of RNase, such as plasmid
preps, from RNA work area.
• It is necessary to use an apparatus that is free of RNase. Particularly avoid any apparatus that has been
used for analysis of plasmid minipreps, since these usually contain copious amounts of RNase. Water
used for making running buffer and staining solutions should be RNAse free.
Native Agarose Gels
The following protocol assumes a standard horizontal electrophoresis apparatus with a 7 X 10 cm
submerged gel with wells of a 20-30 µl capacity. Typically 50 ml 1% agarose in 1X TBE will be used, but
when running smaller sized RNA a higher percentage gel may be more useful.
1. Pour a 1% agarose gel in 1X TBE (89mM Tris base, 89mM boric acid, 2mM EDTA). Be careful to use
clean glassware and high-quality agarose. Use sterile filtered TBE made with RNAse-free water.
Optional: Include 0.5µg/ml ethidium bromide in the gel.
Note: For lower background, omit the ethidium bromide from the gel and post-stain.
2. For each lane, mix RNA sample (0.5-2 µg RNA in water or TE) with an equal volume of RNA Sample
Buffer (10% sucrose, 90% deionized formamide, 0.05% bromphenol blue, 0.05% xylene cyanol). For
Perfect RNA Markers, use 2 µl markers, 8 µl TE, and 10 µl RNA Sample Buffer.
3. Heat at 70°C 3 min, cool to room temperature, and load entire sample onto gel.
4. Run gel at 60 V (up to 10V/cm) in 1X TBE for 1 hour. Monitor progress by the mobility of the dyes in
the sample buffer; the bromphenol blue should be approximately 2/3 down the gel.
5. Stain in 0.5 µg/ml ethidium bromide for 10 – 20 min, destain for 10 – 20 min, and photograph under
UV illumination.
Note: Omit staining if ethidium bromide was included in the gel. An alternative stain (SYBR Green II, Molecular Probes), can be used. Follow the manufacturer’s instructions for SYBR Green II.
Formaldehyde Agarose Gels
A key to good formaldehyde gels is to prepare a thin gel (~2-3 mm). As a guideline, use 1/2 the amount of
gel solution usually prepared for a given apparatus when running DNA. The gel should be pre-
electrophoresed before loading and the buffer recirculated during electrophoresis. The following protocol
assumes 50 ml gel solution (2% agarose in 1X MOPS-EDTA, 2 M formaldehyde) for a standard 10cm ×
15cm gel tray.
Note: 10X MOPS-EDTA = 0.5M MOPS pH 7.0, 10mM EDTA
1. Add 1g high quality agarose and 5 ml 10X MOPS-EDTA to 36 ml nuclease-free water. Boil until
completely dissolved. Pour the hot solution into a sterile RNase-free 50 ml graduated cylinder and add
nuclease-free water to bring volume back to 41 ml to compensate for evaporation. Allow solution to
cool to about 60°C, add 9 ml 37% formaldehyde (12.3 M), and pour the gel in a fume hood. Allow the
gel to stand for 1 hour at room temperature. Place gel in electrophoresis chamber, cover with 1X
MOPS-EDTA and pre-electrophorese at 60V for 30 min.
2. For each lane, mix 3 µl RNA sample (2 – 6 µg RNA in water or TE) with 1.5 µl 10X MOPS-EDTA,
3 µl 12.3M formaldehyde, 7.5 µl deionized formamide. Heat at 70°C for 5 min, place on ice, add 5 µl
RNA sample buffer, mix, and load gel.
3. Run gel at 60V for 1 hour. (BPB dye is approximately 2/3 down the gel.) It is best to recirculate the
running buffer. If recirculation is not available, stop the gel after 30 min and manually mix the buffer
by pipetting from one side to the other.
4. Stain gel for 2 min in nuclease-free water containing 5 µg/ml ethidium bromide. Destain gel in water
for 5 min. Change water and destain another 10 min. Photograph gel or destain more if necessary. Note: Formaldehyde gels are notoriously difficult to stain with ethidium bromide, since dye appears to bind to the gel matrix and cause high background. The background can be reduced by staining
solution less than 5 min and destaining in water. Alternatively, gel can be soaked in water for 10-
15 min prior to staining to remove formaldehyde and allow RNA to renature, thus increasing its
ability to bind ethidium bromide. An alternative stain (SYBR Green II, Molecular Probes) appears
to give superior results for staining RNA. Follow manufacturer’s instructions.
Polyacrylamide-Urea Gels
Polyacrylamide-urea gels provide high resolution of RNA up to 1000 nt in size. The following protocol
assumes 20 ml of gel solution (5% polyacrylamide, 7 M urea gel in 1X TBE) for a standard gel apparatus.
1. Mix 2 ml 50% acrylamide (47.5% acrylamide,
2.5% bis-acrylamide), 14 ml 10 M urea, 2 ml 10X TBE
0.2 ml 10% ammonium persulfate (freshly prepared), 1.8 ml nuclease-free water.
2. Mix and add 10 µl TEMED. Mix again and quickly pipet into gel frame. Insert the comb into position
and allow the gel to polymerize for at least 1 hr. Remove comb, set gel in apparatus with 1X TBE
running buffer, rinse wells with 1X TBE immediately before use.
3. For each lane, mix RNA sample (1-4 µg RNA in water or TE) with an equal volume of RNA Sample
Buffer (10% sucrose, 90% deionized formamide, 0.05% bromphenol blue, 0.05% xylene cyanol). For
Perfect RNA Markers, use 2 µl markers, 8 µl TE, and 10 µl RNA Sample Buffer.
4. Run gel at 150-200 V in 1X TBE running buffer until BPB is approximately 2/3 down the gel.
5. Stain gel in 0.5 µg/ml ethidium bromide for 10-20 min and photograph under UV illumination
(305nm).
References
1. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K.
(1989) in “Current Protocols in Molecular Biology”, John Wiley & Sons, New York.
2. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) in “Molecular Cloning: a Laboratory Manual”,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.。