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The best method is the crush and soak method. It can be sued to isolate single-stranded RNA from denaturing gels. The RNA is of high purity but the method is long and inefficient if long transcripts are to be recovered (cf. less than 30% of DNA greater than 3 kb is recovered).
Protocol
Reagents
Ethidium bromide 10 mg / ml in water Elution buffer 80% formamide in 40 mM PIPES, 1 mM EDTA and 400 mM NaCl (10 mM DTT in water if 35S-labelled RNA is being recovered) Storage buffer 10 mM DTT in sterile water 8M LiCl Isopropanol 70% ethanol
Equipment
Polyacrylamide gel electrophoresis
Methods
In advance
1 Perform polyacrylamide gel electrophoresis as described in m7aiii
Detecting the RNA of interest
Non-radiolabelled fragments
2 Take the gel-plates out of the electrophoresis chambers and lay them flat - they may be quite hot. If only the front plate has been siliconised, place the notched back plate uppermost before separating. The gel should stay attached to the front plate. Insert a metal spatula between the two plates and carefully prise apart. If the gel is sticking mainly to the back-plate (not cleaned well enough!), invert the plates and try again. The gel should remain stuck to one of the glass plates as a support 3 When the plates have been parted, lift the gel onto a piece of Whatmann 3MM paper or equivalent. Lay the glass-plate on a horizontal surface with the gel facing up and lift onto a piece of damp Whatmann paper1 2-3 cm larger than the gel. Lay the paper carefully onto the gel surface, avoiding bubbles or wrinkles. Roll these out with a glass rod. Smooth the paper onto the gel to make the two stick together. Lift one corner of the paper : the gel should stick to it and lift off the glass plate. Remove the paper and gel smoothly in a single motion and then immerse both in a shallow bath of 0.5 mg/ml ethidium bromide in 1 x TBE. Stain for 15 - 45 minutes and remove both the gel and paper2 4 Place a sheet of glad wrap over surface of a UV transilluminator and lie the gel face down on it. Peel the Whatmann paper off and transilluminate with UV light 5 Identify the band and excise it through the glad wrap 6 Crush the slice against the wall of a microfuge tube and cover with 1 - 2 volumes of elution buffer (up to 0.5 ml). Incubate with gentle agitation at 37oC. Fragments < 500 bp will elute in 3 - 4 hours, larger ones will take up to a day 7 Spin briefly to pellet the polyacrylamide then rinse the pellet with 0.5 volumes of elution buffer 8 Precipitate with one volume of isopropanol and recover the RNA by centrifugation. Even small amounts of RNA precipitate very efficiently - due to contaminating polyacrylamide ? - so there is no need to add any carrier RNA 9 Wash the RNA pellet in 70% ethanol and resuspend in 200 ml of 10 mM DTT (with RNasin if paranoid). Add 0.1 volumes of 8M LiCl and re-precipitate with an equal volume of isopropanol 10 Recover by centrifugation, wash the RNA pellet in 70% ethanol and resuspend in 10 mM DTT 11 Quantitate the RNA by UV-induced ethidium bromide fluorescence, by UV absorbance readings or by polyacrylamide gel electrophoresis on an aliquot estimated to contain 50 ng of RNA. Compare with a known standard RNA 12 Store the sample at -70oC in 10 mM DTT
Radiolabelled fragments
13 Prise the gel plates apart as in 2 above and cover the gel surface with glad wrap 14 Mark the glad wrap with a Texta pen to orientate left and right sides of the gel. Expose to X-ray film by placing the film on the bench in a dark room and the gel on its supporting plate immediately on top of it. Expose for 1 - 2 minutes in the dark and then flash the film through the glass plate. Develop the film : the markings on the glad wrap will appear translucent compared to the grey background of the film, and successfully labelled RNA will be apparent as intense black bands 15 Place the film on a transilluminating visible light source and line up the gel on its support plate using the markings on the glad wrap and the outlines of the wells. Excise the band through the glad wrap and then elute as in 5 - 12 above3,4
References
Reference #143 J. Sambrook, E. F. Fritsch and T. Maniatis (1989) Molecular Cloning, A Laboratory Manual.
Reference #151 L. G. Davis, M. D. Dibner and J. F. Battey (1986) Basic Methods in Molecular Biology.
Reference #489 D. E. Titus (1991) Promega Protocols and Applications Guide, Second Edition.
Reference #6 D. A. Melton, P. A. Krieg, M. R. Rebagliati, T. Maniatis, K. Zinn and M. R. Green (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridisation probes from plasmids containing a bacteriophage SP6 promoter Nucleic Acids Res 12:7035-7056
Reference #7 P. A. Krieg and D. A. Melton (1987) In vitro RNA synthesis with SP6 RNA polymerase Methods Enzymol 155:397-415
Reference #12 K. Zinn, D. DiMaio and T. Maniatis (1983) Identification of two distinct regulatory regions adjacent to the human b-interferon gene Cell 34:865-879
This page is maintained by Beverly Faulkner-Jones (b.jones@anatomy.unimelb.edu.au) using HTML Author. Last modified on 10/25/95. 1 Wet evenly with distilled water and blot off the excess with paper towels 2 Handle with gloves - EtBr is carcinogenic 3 Radiolysis of 32P-labelled cRNA is rapid : keep for RNase protection assays for no more than 1 day after purification 4 Purified cRNA for northern hybridisations will keep a few days at -70oC