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Denaturing polyacrylamide gel electrophoresis of RNA



Potassium hydroxide, acetone, ethanol, methanol and Sigmacote / siliconising solution for cleaning and preparing glass gel plates. 40% acrylamide solution 38% w/v acrylamide, 2% w/v bis-acrylamide dissolved in water. Filter through a 0.2 mm filter and store in dark glass at room temperature 1,2,3 10 x TBE 890 mM Tris-borate, 20 mM EDTA, pH ~ 8.3. Add 54g of Tris base, 27.5 g of boric acid and 20 ml of 500 mM EDTA pH 8.0 to 400 ml of distilled water. Make up to 500 ml, filter through a 0.2 mm filter and store at room temperature 4 Urea 5 10% w/v ammonium persulphate solution in water6 TEMED solution7 Formamide loading buffer 80% formamide8, 1 x TBE, and 1 mg / ml bromophenol blue


Glass plates Vertical electrophoresis tank Gel combs and spacers Metal plate.


Preparation of glass plates

1 Select a pair of glass plates of the appropriate size - long for increased resolution and wide for a large number of samples - and spacers and a gel comb. Make sure the spacers and comb are exactly the same thickness9, 10,11,12 2 With new or dirty plates, wipe the surface with KOH in methanol (5 g / 100 ml) and rinse off in distilled water until all the KOH in methanol is removed. If the plates are not too dirty, acetone alone may suffice. Handle the glass plate by the edges only and clean first with 70% ethanol, then 100% ethanol and finally 100% methanol, using a soft cloth. Allow the plates to dry before siliconising the front-plate (no rabbit-ears) with Sigmacote or any other glass-siliconising solution. Spread a few drops evenly over the surface with a soft cloth. Repeat until the friction between plate and cloth it is minimal, allowing the plate to dry in between each application13,14,15,16 3 Clean the spacers and comb with distilled water, ethanol then methanol, and check that they are all compatible when the gel-plates are assembled (but before the gel is poured!). Place the plates together with the side spacers in place and clamp the sides with bull-dog clips. Seal the bottom of the plates twice with tape, taking care with the corners : this is where most gels leak during casting

Casting the gel

4 Decide on the percentage acrylamide gel required. The figures are actually for DNA, but are a guide for RNA :-
% age acrylamide (w/v) Effective range of Size of RNA co-Size of RNA co-
with BIS at 1:20 separation - bpmigrating with Xylene migrating with
Cyanol (33)Bromophenol Blue
3.51 000 - 2 000460100
5.080 - 50026065
8.060 - 40016045
12.040 - 2007020
15.025 - 1506015
20.06 - 1004512
5 ~ 60 ml of acrylamide solution is required for standard sequencing gels. Make up ~ 20 ml for 15 x 15 cm gels
20 ml60 ml
Final % 40% stock 10% TEMED40% stock 10%
acrylamideacrylamideammonium acrylamideammonium
persulphate persulphateTEMED
4%2 ml6 ml
5%2.5 ml7.5 ml
6%3 ml9 ml
7%3.5 ml10.5 ml
8%4 ml200 ml20 ml12 ml600 ml60 ml
9%4.5 ml13.5 ml
10%5 ml15 ml
11%5.5 ml10 g of 16.5 ml30 g of
12%6 mlsolid urea18 mlsolid urea
Add the acrylamide and TBE to a final concentration of 1 x to the urea and make up to the final volume with MilliQ water. Mix on a rotating wheel until all the urea has dissolved 6 Add the ammonium persulphate and mix by inverting the tube 7 Add the TEMED and mix by inverting the tube 8 Draw the solution into a 20 / 60 ml syringe through a wide-bore (18 gauge) needle, then change to a narrow-bore (21 gauge) needle17. Hold the glass plates at approximately 45 degrees to the vertical, and incline to the right by balancing the plates on the bottom right-hand corner. Slowly and continuously inject the acrylamide solution down the right side of the gel, taking great care not to introduce air bubbles. Fill the bottom right-hand corner first, keeping the air-acrylamide interface smooth. Gradually alter the angle of the gel plates whilst still injecting so as to fill across the bottom of the gel. Continue injecting down one side of the gel until the acrylamide reaches the top 8 If there are any air bubbles trapped between the plates, it may be possible to remove them with a long spacer thinner than the ones used to cast the gel18 9 Lay the gel plates horizontally on support and insert the comb, taking care not to introduce bubbles around the teeth. Top up with acrylamide solution. Insert the comb so that the well 'walls' will extend to the top edge of the back (notched) glass plate. Leave to polymerise for 45 - 60 minutes at room temperature : a sharp, straight schlieren line should be visible around the teeth of the comb if polymerisation has occurred properly. The gel should be used immediately for RNA19,20,21,22

Loading and running the gel

10 Remove the comb carefully from the fully polymerised gel - it is safest to do this under buffer with the bull-dog clips still place. Immediately rinse out urea and fragments of un-polymerised acrylamide from the wells with 1 x TBE and a syringe with an 21 gauge needle 11 Remove the sealing tape and then all but the top pair of bulldog clips : take care that the two plates do not move. Clip the plates into a vertical electrophoresis apparatus (notched back-plate against the cathode chamber). 12 Clip the metal plate over the front gel plate23. The gel warms up unevenly during electrophoresis : the centre becomes hotter and the gel 'smiles'. The metal plate causes even diffusion of the heat over the gel plates and ensures even running of the samples. Fill the tank with 1 x TBE buffer and connect to the power supply (black = cathode at the top of the gel and red = anode at the bottom). Pre-electrophorese for 30 - 45 minutes at constant power (40-50 watts for a 20 cm x 40 cm long gel, roughly 1700 V) 13 Resuspend RNA samples in formamide loading buffer. Sharks-teeth combs will take up to 3 ml in volume and the wells in a 0.4 mm thick gel will usually take up to 7 ml. Heat denature the samples in loading buffer for 5 minutes at 70oC and then chill on ice 14 Turn off power to gel and rinse out the wells again with a syringe, until no urea can be seen floating into the cathode buffer. Load the samples carefully into the bottom of the well24 15 Electrophorese at constant power (40-50 watts for a 20 cm x 40 cm long gel, roughly 1700V) for the appropriate time : For a 5 - 8% sequencing-sized gel, it takes approximately 1-11/2 hours for the BPB dye front to run to the bottom (constant power, 45 - 55 watts) Using a 5% gel, to separate 400 bp from 425 bp, the BPB dye front needs to be run off the bottom twice Using a 10% gel, a 77 bp fragment is 1/3 of the way down the gel when the BPB reaches the bottom the first time
% Polyacrylamide gelBromophenol blueXylene cyanol (41)
The sizes are given in base pairs for RNA that co-migrates with the marker dye. RNA of the same molecular weight migrates approximately 5 - 10% slower than RNA (at 40 - 45V/cm). The differences are minimised by running the gel as fast as possible

Detecting RNA in denaturing polyacrylamide gel

Non-radiolabelled fragments
16 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 17 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 paper25 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 paper26 18 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 photograph with UV light and / or excise the band of interest
Radiolabelled fragments - non-fixed gels
19 Prise the gel plates apart as in 16 above and cover the gel surface with glad wrap. Expose the gel to X-ray film. If probes are being made and only a short (minutes) exposure is required, invert the gel on a piece of film in the dark room. For longer exposures (a few hours), use a film-cassette 27
Radiolabelled fragments - fixed gels
20 Prise plates apart as in 16 above28 21 Lay the glass-plate on a horizontal surface with the gel facing up and lift onto a piece of damp Whatmann paper as described in 17 above and lay on a flat surface. Cover the gel in glad wrap and dry on a commercial gel dryer at 80oC for up to 2 hours 22 Remove the dried gel and peel off the glad wrap. The surface should feel smooth but not sticky 23 Autoradiograph or PhosphorImage as desired


1 RNA fragments labelled with 35S generally have to be dried for autoradiography / PhosphorImaging. A wet gel absorbs too much of the signal

2 If the paper refuses to stick to the gel, blot carefully with tissues to remove some of the water and try again

3 If the gel is torn or wrinkles badly during lifting, gentle washing with a distilled water bottle can be used to get it back into place on the paper surface. If this does not work, float the gel in a bath of distilled water and recover again onto wet Whatmann paper. NB, this gel will the take longer to dry than one which has not be immersed after electrophoresis and some of the sample will elute into the water

4 If the paper refuses to stick to the gel, blot carefully with tissues to remove some of the water and try again


Many but contained mainly within :- 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

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This page is maintained by Beverly Faulkner-Jones ( using HTML Author. Last modified on 10/25/95.

1 Acrylamide is a potent, skin absorbed neurotoxin 2 Acrylamide and bis-acrylamide are slowly deaminated to acrylic acid ; the reaction is catalysed by light and alkali. Check the pH of the solution (neutral) and keep it dark and cool. Re-make solutions every few months 3 Cheaper grades of acrylamide often contain contaminants so always use sequencing grade reagents 4 Discard the solution when a precipitate forms 5 Use sequencing grade reagents. 6 Will keep for a few weeks at 4oC 7 Keep at 4oC 8 Deionise the formamide with a mixed bed ion-exchange resin (AG 501-X8 Resin, BioRad, Hercules, CA). If the pH of the formamide after deionisation is>7.4, discard it 9 Thin gels (0.3 mm - 0.5 mm) give better resolution, do not heat up as much and are easier to fix and dry than thick (1 mm) gels. However, they are more fragile, more difficult to cast and don't allow such large amounts of sample to be loaded 10 Conventional well-forming combs can be used but, especially at low acrylamide concentrations, there are frequently problems with tearing and deformation of wells. They give good results when analysing RNase protection assays or purifying probes or nucleic acids 11 Alternatively, sharks teeth combs can be used. These give a flatter, more uniform loading-surface than the well-forming combs and reduce the risk of tearing/damage to the gel. They give good results when analysing sequencing reactions : the close proximity of the lanes allows easier reading of the sequence, but they are prone to allowing leakage of samples between wells 12 The thinner and longer the gel, the more important it is to thoroughly clean and siliconise the plates. Dirty plates will not allow even casting of the gel (air bubbles) and the gel is liable to tear when the plates are separated after electrophoresis 13 If pouring a low-percentage acrylamide gel, casting is easier if the back-plate is also siliconised 14 If a well-forming comb is to be used, do not siliconise the very top of the back-plate : the teeth of the well will collapse if the glass surface is too slippery 15 Plates washed with distilled water after previous electrophoresis just need a methanol wash and re-siliconising 16 Siliconising agents are toxic : use in a fume hood 17 This reduces the likelihood of injecting air bubbles trapped within the syringe 18 Only necessary if the full width of the gel is to be used or when desperate! 19 Gels cast the day before tend to produce smeary RNA bands - ? urea / acrylamide decomposition ? 20 When preparing low-percentage gels with well-forming combs, the wells tend to collapse. Accelerating polymerisation helps - use warmed acrylamide solutions BUT there is no leeway to make mistakes when pouring : the gel sets fast! 21 High percentage gels will polymerise well with half the amount of AP and TEMED and give more time to cast them and make mistakes 22 Allow all unused acrylamide to polymerise - essentially non-toxic - before disposal 23 Not all makes of vertical gel apparatus needs this : many have an integral metal plate positioned against the back (notched) glass plate. Also, it is usually unnecessary for small 15 x 15 cm gels and may not be a problem with a large RNase protection assay gel which will only be run ~15 cm (BPB dye front) 24 There are a variety of methods available. I use tips that are flattened front to back and slip between the two plates if a 0.4 mm spacer is used. A single tip can be used for each gel by washing it out in the anode chamber with 1 x TBE between loadings 25 Wet evenly with distilled water and blot off the excess with paper towels 26 Handle with gloves - EtBr is carcinogenic 27 This is the method to use when radiolabelled-probes are to be recovered from gels. If 32P is used as the radio-label, the gel does not have to be fixed and dried prior to autoradiography, but the resolution is poorer 28 Fixing RNase protection assay gels in methanol / acetic acid reduces the sensitivity of the assay as some of the sample is lost into the fixative