Team:HKUST/Protocols/Agarose gel

Agarose gel preparation and gel electrophoresis

Purpose: To check the result

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:

1. To make a 0.8% agarose gel, use 0.16 g agarose per 20 mL 0.5x TBE.
2. Cover the flask with plastic wrap to prevent boiling over, then microwave the solution for 1-2 minutes to dissolve the agarose.
3. Let agarose solution cool for 5 minutes, then add SYBR safe DNA gel stain to a final concentration of 0.5 μg/mL.
4. Pour the agarose solution into a casting tray with well comb in place. Allow 20-30 minutes to completely solidify.
5. Place the agarose gel into the gel box and fill the box with 0.5X TBE until the gel is immersed.
6. 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.
7. Add 1 μL loading dye per 5 μL of sample.
8. Load the samples from left to right.
9. Cover the gel box and plug in the electrodes in a way that the samples will run towards the positive, red electrode.
10. Run the gel at 100V until the dye line is approximately 50-75% of the way down the gel. Approximately 20-30 minutes.
11. Carefully remove the gel from the gel box and check the result under UV exposure.

Tips:

A. Higher concentration of agarose solution makes better resolution for less molecular weight expected band.
B. Let bottom of the flask be immersed in a cup of cold water for faster cooling.
C. 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:

1. Be sure to wear a glove before treating the hot flask.

2. Ethidium bromide is a strong mutagen. Wear a lab coat, eye protection, and gloves when working with this chemical.

• Agarose gel preparation and gel electrophoresis
• 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