Team:HKUST/Protocols/Agarose gel

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'''Agarose gel preparation and gel electrophoresis'''  
'''Agarose gel preparation and gel electrophoresis'''  
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**Be sure to wear a glove before treating the hot flask.
**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.
**Ethidium bromide is a strong mutagen. Wear a lab coat, eye protection, and gloves when working with this chemical.
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel
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electrophoresis</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li>
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Revision as of 08:59, 18 October 2009

Salt and Soap template

a

'''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: **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.
HKUST

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