IGEM:British Columbia/Protocols/Gel Verification

Gel Verification

Materials

• Agarose (1g/100mL for a 1% gel)
• 0.5X TBE Buffer (dilute 10X TBE into diH2O)
• Loading buffer
• SYBRSafe
• 1/20 dilutions of 100bp or 1kb ladder

Equipment

• Gel casting trays, combs, and box

Method

• Weigh out suitable amount of agarose into an Erlenmeyer flask. (usually 0.5g for 50mL 1% gel)
• Add ~1/2 the final volume of 1X TBE.
• Plug flask lightly with a Kimwipe or punctured parafilm, then warm in microwave. Remove when large bubbles start forming.
• Add the remainder of the TBE. If still hot, cool under running water.
• Add SYBRSafe (1/10000, e.g. 5uL/50mL) and swirl to mix.
• Pour into gel casting tray and insert comb. Protect from light.
• While cooling, prepare samples by adding loading buffer to appropriate final concentration.
• After gel has solidified, remove from tray and place in gel box. Fill box with 1X or 0.5X TBE (if only taking a picture of the gel, TBE can be reused 2 or 3 times, but use new TBE if performing gel extraction).
• Load samples and ladder into wells.
• Run gel until dye is 3/4 off the gel, approx 40 minutes at 100V
• Remove gel and place on transilluminator/imager. Take picture.
• Cross-reference sample bands with ladder bands to determine band size.