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Preparation and Examination of
Reagents1 20 x MOPS buffer 400 mM 3-(N-morpholino)propanesulphonic acid, 160 mM sodium acetate, 20 mM EDTA, pH 7.0. Sterilise by autoclaving2 Ethidium bromide 10 mg / ml in sterile MilliQ water Agarose Formaldehyde3 RNA loading buffer 1 x MOPS buffer (ie, 20 mM MOPS, 8 mM sodium acetate and 1 mM EDTA, pH 7.0), 7% (w/v) formaldehyde pH 4.0, 5% (v/v) sterile glycerol, 50% (v/v) deionised4 formamide and 0.025% (v/v) of saturated aqueous bromophenol blue solution 5 RNA samples
Electrophoresis power supply
Preparation of agarose gel 1 Clean the gel-former and comb with distilled water, then 70% ethanol. Seal the edges of the gel-former with tape. Check that the comb sits approximately 1 mm above the gel-former when in situ 2 Make up enough buffer for both the electrophoresis tank and the gel to avoid any differences in ionic strength between them 3 For 100 ml of a 1.3% gel : melt 1.3 g of agarose in 50 ml water. Add to 20 ml of formaldehyde 6, 5 ml of 20 x MOPS and 5 ml of ethidium bromide solution in a measuring cylinder. Make the volume up to 100 ml with MilliQ water 4 Swirl to mix and then pour the gel carefully, checking for air bubbles under or between the teeth7. The final gel should be between 3 mm and 5 mm thick. Allow it to set at room temperature for 30 - 45 minutes 8, 9, 5 When fully set, pour enough buffer to cover the gel surface by ~ 1 mm and allow to stand for a couple of minutes 10. Remove the comb carefully to avoid tearing the bottom of the wells (and subsequently losing the sample), and place the gel in the electrophoresis tank. Cover to a depth of ~ 1 mm
Prepare RNA samples 6 Prepare the RNA to be analysed. The loading buffer can be diluted by up to 2 fold although generally 5 ml of aqueous RNA is added to 15 ml of RNA loading buffer11 7 Denature the RNA samples in loading buffer by heating to 70oC for 5 minutes 8 Chill on ice before loading the gel 12, 13, 14,
Electrophoresis of RNA sample 9 Load the RNA samples carefully into the slots. Connect the electrophoresis tank to a constant voltage power supply - RNA will run from black to red ie cathode to anode : make sure leads are on the right way round ! Run at 1 - 5V / cm (measured as the distance between the electrodes) until the BPB dye front has migrated the appropriate distance. Check after the BPB has run 50% of the way down the gel. The ethidium bromide will migrate the opposite way to the RNA and long electrophoresis will remove much of the ethidium from the gel15
Examination of RNA in agarose gel 10 This relies on the UV-induced fluorescence of intercalated ethidium bromide. However, formaldehyde fluoresces brightly when irradiated with UV light at 320 nm. Remove the formaldehyde by immersion in MilliQ water - up to 6 changes over up to 2 hours16 11 For a permanent record, photograph the gel when trans-illuminated by a 302 nm wavelength UV light source. This wavelength causes fluorescence of the intercalated ethidium, but reduces the amount of 'UV nicking' of the RNA. Both ethidium fluorescence and RNA nicking are maximal at 254 nm.
References Reference #143 Tan Lab Library 07-94> Sambrook J, Fritsch EF, Maniatis T. 1989 Molecular Cloning, A Laboratory Manual, Second Edition. Cold Spring Harbour Laboratory Press. Reference #151 Tan Lab Library 07-94> Davis LG, Dibner MD, Battey JF. 1986 Basic Methods in Molecular Biology. Elsevier. New York
Reference #5 J. D. Allen, T. Lints, N. A. Jenkins, N. G. Copeland, A. Strasser, R. P. Harvey and J. M. Adams (1991) Novel murine homeo box gene on chromosome 1 expressed in specific haematopoietic lineages and during embryogenesis Genes Dev 5:509-520
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1 All reagents are made up in sterile MilliQ water unless otherwise stated 2 The solution turns yellow but this does not seem to interfere with its performance 3 Use saturated formaldehyde solution - Analar or equivalent. The concentration is ~37% in water and the pH should be <4.0. If the pH is>4.0 or if there is a lot of yellow 'sludge' on the bottom of the container, get fresh stocks 4 Formamide was routinely deionised using a mixed bed ion-exchange resin (AG 501-X8 Resin, BioRad, Hercules, CA). If the pH of the formamide after deionisation was>7.4, it was discarded 5 Make up saturated BPB solution in sterile MilliQ water 6 This is ~7.4% formaldehyde or ~2.4M. This can be reduced to ~4% / 1.2M unless the RNA of interest is particularly large 7 The gel will be ~60oC after addition of ~50 ml of reagents at room temperature 8 A paper tissue held over the mouth of the flask will catch bubbles / particle of partially dissolved agarose 9 Setting of the gel can be accelerated by placing the former in a cold room 10 This helps to free the teeth of the gel comb and prevents them tearing the gel when the comb is removed. This should always be done for low percentage and low melting point agarose gels 11 There are four common gel loading buffers which are all 6 x concentrates : I = 0.25% BPB, 0.25% XC in 40% w/v sucrose; II = 0.25% BPB, 0.25% XC in 15% w/v Ficoll 400; III = 0.25% BPB, 0.25% XC in 30% v/v glycerol and IV = 0.25% BPB in 40% w/v sucrose 12 Using too large a sample volume in the slot can result in contamination of adjacent lanes. 13 Marker RNAs may be used 14 If the RNA sample contains any alcohol, the RNA will 'creep' out of the well after loading - heat to 70oC for 10 minutes in an open tube before adding the loading buffer 15 The gel may be stained in ethidium bromide solution after electrophoresis is complete. Immerse in 0.5 mg / ml ethidium solution for ~30 - 45 minutes at room temperature 16 Formaldehyde should be difficult to smell in an adequately washed gel