Preparationofdenaturing6％

Preparation of denaturing 6% polyacrylamide gels for microsatellite analysis

(also for SSAP, high-resolution IRAP/ISSR and other analyses)

Saadiah Jamli and Pat Heslop-Harrison November 2003

University of Leicester

Preparation of the plates

Bigger plate for treatement with Repel-silane

a) Wear gloves. Clean the plate with laboratory detergent (type used for cleaning radioactivity – e.g. 25% Lipsol) and warm water. Rinse.

b) Clean the upper surface with 100% ethanol. Using blue rolls of tissues, polish until it ‘squeaks’.

c) Apply a few drops of Repel-silane to the upper surface of the plate and spread evenly using blue roll.

d) Wipe with warm water and then 100% ethanol, and let air dry.

Change gloves between working with Repel-silane and Bind-silane

Eared plate for binding

a) Clean the plate with detergent (25% Lipsol) and warm water. (You may also need to use a razor blade to remove old bits of gels that have stuck). Rinse.

b) Clean the upper surface with 100% ethanol. Using blue roll, polish until it squeaks.

c) Apply 20 ml Bind-silane to the upper surface of the plate and spread evenly using blue roll.

d) Wipe with warm water and then 100% ethanol, and let air dry.

Ensure the spacers are clean and dry. Align and sandwich between square plate and the eared plate. Secure plates with bulldog clips.

Preparation of polyacrylamide gel

With gloves, prepare

4.5 ml acrylamide solution (care – neurotoxin)

3.0 ml 10X TBE

14.4 g urea

Top up with deionized water to 30 ml for 20cm x 20cm plates.

De-gas this mixture (evacuate 2-3 times in a vacuum desiccator).

Add 15 ml TEMED to the solution.

Add 150 ml 10% fresh ammonium persulfate (or from aliquots frozen at –20C) and swirl around.

Working quickly (maximum 5 min), pour the gel mix on square plate carefully to avoid bubbles, while sliding in the eared plate (or use a 50ml syringe).

Insert comb into to of plate: if using sharkstooth/sawtooth comb, place flat side against the top of the gel, displacing some liquid.

Leave to polymerise for about 1 hour.

Remove top spacer, and wash well with distilled water from a wash bottle to remove both unpolymerized acrylamide and crystallized urea. This is very important. Wash bottom of gel to removed unpolymerized acrylamide.

Polyacrylamide gel running

Pour 1X TBE into the top reservoir and lower reservoir of the gel apparatus.

Pre-run at 25 watts for 30 minutes to ‘clean’ and pre-heat gel.

Meanwhile denature the PCR reaction for 5 minutes at 95OC and straight away place on ice.

Load the sample.

Marker ladder: 5 ml hyperladder + 3 ml formamide loading buffer

PCR reaction: 3 ml PCR product + 3.0 ml formamide loading buffer

Load 6 ml of each sample into individual wells of the gel

Run gel until the dark blue just runs off the bottom of the gel or as appropriate.

Silver staining

a) Fixer (10% acetic acid)

Add 50 ml glacial acetic acid to 450 ml distilled water

b) Silver stain

Add 3 ml 1N silver nitrate solution in 500 ml distilled water.

Then add 0.75ml formamide

c) Developer

Dissolve 15 g sodium carbonate in 500 ml distilled water and put at 4OC

1. Remove the gel and separate the plates carefully with a single-edged razor blade.

2. Place the gel in tray with the fixer and leave shaking in a fume hood for 30 minutes. Pour off fixer (save).

3. Wash with water for 10 – 15 minutes, on shaker. Rinse again and pour off the water.

4. Add silver-stain and leave shaking for 30 minutes (Silver stain can be re-used up to 10 times).

5. Immediately before developing the gel, add in 75 ml sodium thiosulphate solution (0.1N) and 0.75 ml formamide to the pre-chilled developer solution.

6. After silver-stain (step 4), agitate the gel several times for 10 seconds.

7. Immediately agitate the gel in the developer until band development progress sufficiently.

8. Stop the reaction by adding the fixer saved earlier (step 2). Agitate until bubbles cease.

9. Rinse gel in water for 20 minutes.

10. Leave to surface dry by standing vertically for 10 min.

11. Scan the gel with a computer flatbed scanner, or photograph.