Team:HKUST/Protocols/Agarose gel

Agarose gel preparation and gel electrophoresis

* *Materials: Agarose, 0.5X TBE, SYBR safe DNA gel stain, DNA loading dye, DNA ladder/marker **Choice of ladders: VC100bp Plus DNA ladder (highest band 3000bp); Lamda DNA BstEII marker (highest band 8400bp, 1% argarose) *Procedure: **To make a 0.8% agarose gel, use 0.16 g agarose per 20 mL 0.5x TBE. **Cover the flask with plastic wrap to prevent boiling over, then microwave the solution for 1-2 minutes to dissolve the agarose. **Let agarose solution cool for 5 minutes, then add SYBR safe DNA gel stain to a final concentration of 0.5 μg/mL. **Pour the agarose solution into a casting tray with well comb in place. Allow 20-30 minutes to completely solidify. **Place the agarose gel into the gel box and fill the box with 0.5X TBE until the gel is immersed. **Load an appropriate molecular weight ladder into the first lane of the gel. If needed, an additional ladder with different molecular weight could be loaded into the last lane. **Add 1 μL loading dye per 5 μL of sample. **Load the samples from left to right. **Cover the gel box and plug in the electrodes in a way that the samples will run towards the positive, red electrode. **Run the gel at 100V until the dye line is approximately 50-75% of the way down the gel. Approximately 20-30 minutes. **Carefully remove the gel from the gel box and check the result under UV exposure. *Tips: **Higher concentration of agarose solution makes better resolution for less molecular weight expected band. **Let bottom of the flask be immersed in a cup of cold water for faster cooling. **In order for clearer band to cut specific band on the gel, immerse the gel in Ethidium bromide for 10-15 minutes after step k. *Safety tips: **Be sure to wear a glove before treating the hot flask. **Ethidium bromide is a strong mutagen. Wear a lab coat, eye protection, and gloves when working with this chemical.

• Purpose: To check the result
• PCR (Taq, Vent)
• PCR product clean-up
• Enzyme digestion (vector, insert)
• Plasmid DNA extraction
• Gel cutting
• Gel extraction
• Ethanol precipitation
• Ligation
• Transformation to E.coli
• Transformation to yeast
• Cell lysis
• Yeast genomic DNA extraction
• Western blotting
• GFP testing
• Biological test