MadgeBio Ltd.

PROTOCOL FOR MAKING AND RUNNING MADGEBIO GELS

1. (M0111, M0131, M0141, M0151 and M021 H-Page)

2. PREPARING MADGEBIO GELS. Silanise a glass plate on one surface. Spread 1ml of Sticky Silane (MR0999) as evenly as possible. Spreading the liquid with the edge of another glass plates ( MA0012) dose this well, care must be taken to spread the sticky silane to the edges of the plate. When dry, wipe surface with a wetted tissue. (If using the MadgeBio gasket for application not needing the glass support omit this step.)

3. Lay the MadgeBio gel former level and with teeth upward on paper towel or in a flat tray. Please ensure the lip on the short edge is away from you.

4. Mix your usual gel mix and pour into the nearest end of the MadgeBio gel former. Using 3x excess TEMED and APS will speed the setting of acrylamide gels, to as little as 10 minutes.

5. Lay the glass plate silanised face down (toward the teeth). There are two accepted methods to cast the gels, a) put one end of the glass in place and use one smooth, continuous action to lower the other end down, or b) place the edge of the glass plate on the front edge of the former and slide the plate over the former, in a manner similar to that used when casting a sequencing gel. Once in place, place a 50-100 g weight, e.g. a bottle, centrally on the glass. DO NOT lift the glass again or your may introduce air bubbles.

6. Once the MadgeBio gel has set, use a spatula to prise the glass plate with the gel attached, away from the former. Using a flat ended spatula under the centre of the far edge of the glass, where there is a small gap. This will laeve a slightly jagged edge t your gel.

7. RUNNING MADGEBIO GELS. Place the gel in the MadgElectrophoreseW gel tank, (M0054), this has a special adapter (M0154) inside to ensure that buffer effects found in larger gel tanks are removed.

8. SAMPLE PREPARATION. Away from the gel tank, add 2ul loading dye to 5ul PCR sample. This can easily be carried out with a multi-channel pipette, or if there is high throughput with a RepMate or a Robot.

9. LOADING GELS. Load 5ul (or suitable volume) using 8, 12, channel pipette or using the MadgeBio RepMate, (REP0001, REP0002, REP0005), passive replicator. If using pipettes loading can be done under buffer. Loading gels away from the gel tank may be advantageous, ensure the wells are full of buffer before loading. Then carefully lower the gel into the tank with the glass plate over it. Gels can easily be stacked and placed in a gel tank in this way. (Loading with a RepMate see Tech Note Rep003) Conversely gels can be run "semi dry", place a single gel in the gel tank, and fill the buffer up to the level of the gel, do not fill above level of the top of the gel.

10. Typically running conditions would be 10V/cm for 30 minutes, in the MadgElectrophoreseW this is an approximately 170 volts. But running conditions should be adapted to suit your needs and applications, we have run for 10 minutes and achieved excellent resolution of DNA fragments.

11. VISUALISATION AND ANALYSIS OF GELS, Stain the gel with Ethidium Bromide. Pre-Staining leads to less diffuse bands than post-staining , however both methods give acceptable results. (Please be consistent with method of staining, as samples will run differently between Pre- and Post- stained gels) Pre-stain in a staining tray on a rotory shaker for 30 minutes, this is usually sufficient. The use of Vistra Green, SyBr Green or a similar stain also gives good results, however you should bare in mind that gels containing these dyes cannot be stored for any length of time, as the dye deteriorates.

12. Image capture may be carried out on any system that offers a TIFF output, that is for example any CCD camera, FluorImagers etc.. Automatic analysis software (MS0002 and MS0003) is available to intereprete the image of the array, this leads to rapid and easy analysis of the gel. It will additionally lead to rapid transfer of data to a database, such as Excel, with the need of only 2 keystrokes.



PROTOCOL FOR LOADING MADGE GELS WITH REPMATES

1. Place pins of the RepMate (REP0001, REP0002, REP0005) in wells of the PCR plate, raise and lower replicator pins several times to load the slots. The top of the slots must be submerged under the level of the liquid. If the liquid level in the wells is lower than the height of the slot, move the slot pins to the edge of the well to create a capillary effect between the pin and the well to fill the slot. If the volume in the well is very low (50 ul or less), then you must pre-wet the slot pins in a sterile water bath that is deeper than the top of the slot. Next blot on to a sterile pad ( Lint Free Blotting Paper - M0092) to empty the slot and then place the pre-wetted slot pins into the source wells and allow capillary action to fill the slots. ( Use of the Madge Surfactant (MR0091) will assist in the consistent wetting of the probes)

2. Move the replicator in a slow circular motion wiping the pins against the well walls as you raise the slot pins from the wells. This reduces the amount of liquid hanging on the sides of the pins. The liquid in the slot can be delivered to a MADGE gel, containing running buffer, another microplate containing liquid or to a membrane or other absorbent surface.

(Note: The liquid to be transferred must contain a surfactant such as a detergent, protein, DNA, carbohydrate, etc. to reduce surface tension. Using water and dye alone will result in inadequate loading of the slots and should not be used to test the system)

3. Place the Template on a flat surface, the top of a polystyrene enzyme box brings the template to a good height, for easy manipulation of the RepMate. This is totally dependent upon height and technique. Ensure the text is facing you and readable.

4. Place the gel, support side down, with the extreme upper left and extreme lower right marker wells over the red marker wells on the template.

5. Take the RepMate and place the front 12 transfer probes in the front 12 wells on the gel. A purposeful movement backwards allows the remaining 84 probes to go in to the wells in a single action.

6. Leave the Repmate in the wells for 5-10 seconds and then remove carefully from the gel. Making sure that sample does not spill onto the top of the gel. This is no problem in Acrylamide gels, the base of the well is glass - with Agarose care must be taken to NOT break the agarose in the base of the well - the replicator must be held off of the base manually.