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m7aiii
Protocol
Reagents
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
Equipment
Glass plates
Vertical electrophoresis tank
Gel combs and spacers
Metal plate.
Methods
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 - bp | migrating with Xylene | migrating with |
| | Cyanol (33) | Bromophenol Blue |
| 3.5 | 1 000 - 2 000 | 460 | 100 |
| 5.0 | 80 - 500 | 260 | 65 |
| 8.0 | 60 - 400 | 160 | 45 |
| 12.0 | 40 - 200 | 70 | 20 |
| 15.0 | 25 - 150 | 60 | 15 |
| 20.0 | 6 - 100 | 45 | 12 |
5 ~ 60 ml of acrylamide solution is required for standard sequencing gels. Make up ~ 20 ml for 15 x 15 cm gels
| | 20 ml | | | | 60 ml | |
| Final % | 40% stock | 10% | TEMED | 40% stock | 10% | | |
| acrylamide | acrylamide | ammonium | | acrylamide | ammonium | | |
| | persulphate | | | persulphate | TEMED | |
| 4% | 2 ml | | | 6 ml | | | |
| 5% | 2.5 ml | | | 7.5 ml | | | |
| 6% | 3 ml | | | 9 ml | | | |
| 7% | 3.5 ml | | | 10.5 ml | | | |
| 8% | 4 ml | 200 ml | 20 ml | 12 ml | 600 ml | 60 ml | |
| 9% | 4.5 ml | | | 13.5 ml | | | |
| 10% | 5 ml | | | 15 ml | | | |
| 11% | 5.5 ml | | 10 g of | | 16.5 ml | | 30 g of |
| 12% | 6 ml | | solid urea | | 18 ml | | solid 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 gel | Bromophenol blue | Xylene cyanol (41) |
| 5 | 35 | 130 |
| 6 | 26 | 106 |
| 8 | 19 | 76 |
| 10 | 12 | 55 |
| 12 | 8 | 28 |
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
Notes
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
References
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 (b.jones@anatomy.unimelb.edu.au) using HTML Author. Last modified on 10/25/95.