SSCP gel preparation:
Assemble the gel casting apparatus first. Clean the casting glass with distilled water followed by ethanol to remove any grease from the surface.
Pour a few drops of melting point bath oil onto the glass plate with sample wells mould and equally wipe the glass with Kimwipes. Make sure there is no tissue fibre left on the glass. Pre-wet the other glass plate with distilled water first, then mount a piece of GelBond PAGfilm onto the glass. Clamp the two glasses tightly and ensure that there’s no leakage during gel pouring.
Prepare the SSCP gel as follow:
Distilled water 10mL
*Bis-Sep 8.3m
**Gel Buffer 2.2mL
APS (40%, w/v) 60mL
TEMED 40mL
*It consists of 5.8g acrylamide and 0.2g bisacrylamide which dissolved in 20mL
distilled water).
**It consists of 4.36g Tris in 100mL distilled water, pH8.4.
Mix the solution thoroughly and pour it into the gel casting apparatus immediately. Make sure that there is no bubble trapped within the gel. Let the gel polymerized for 15-20 minutes at room temperature.
Pre-set the electrophoresis unit to 15°C. Place the polymerized gel in the electrophoresis unit until use.
Sample preparation and gel electrophoresis:
Denature the sample with *denaturing solution in 1:1 ratio at 95°C for 5 minutes. Chil on ice for another 5 minutes.
*It consists of 99% formamide, 1% xylene cyanol solution and tiny amounts of bromophenol blue.
2. Load the denatured sample onto the polymerized gel. Start electrophoresis at 200V for about 2-3 hours at 15°C.
SSCP Gel staining:
After electrophoresis, stain the gel with commercial silver staining kit, or the home made reagents.
Fix the gel first with fixing solution for at least 30 minutes at room temperature with gentle shaking.
Stain the gel with staining solution for at least 30 minutes at room temperature with gentle shaking.
Incubate the gel with distilled water for 1 minute at room temperature with gentle shaking.
Develop the gel with developing solution for about 6 minutes at room temperature with gentle shaking.
Stop the developing reaction with stopping solution for at least 30 minutes at room temperature with gentle shaking.
Rinse with distilled water for at least 30 minutes at room temperature with gentle shaking.
Transfer the gel onto the blotting paper to remove excess water. Wrap the gel with Saran Wrap.
