McClean: Agarose for gels

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Contents

Overview

Protocol for making gels for running out and visualizing DNA. You should choose the percentage of gel based on the size of fragments you would like to separate. Commonly, percentages between 0.7% and 2% are used. Large fragments (5-10kb) will show good separation (resolution) on a 0.7% gel and small fragments (0.2-1kb) will show good separation on a 2% gel. We almost always use 1% gels.

CAUTION: Ethidium bromide is a potent mutagen and is an irritant to the eyes, skin and respiratory tract. Ethidium bromide can be absorbed through exposed skin and mucus membranes. Always wear appropriate protective equipment when working with ethidium bromide: nitrile gloves, a lab coat, and safety goggles.

Materials

  1. Agarose (Fisher BP1356)
  2. TAE Buffer (1x)
  3. Ethidium bromide (1% solution = 10mg/ml, Fisher BP1302)
  4. "Hot-Hands" (for removing agarose from the microwave)

Procedure

  • Weigh out 0.5 g of agarose into a 250 mL conical flask. Add 50mL of 1x TAE , swirl to mix. This is for a 1% gel (1g/100ml buffer). Use as large a container as possible, because the agarose boils over easily.
  • Microwave for about 1 minute to dissolve the agarose, but monitor to make sure the agarose doesn't boil over. Swirl occasionally to avoid superheating the solution. Wear gloves and use hot-hands!
  • Use the silicone "hot-hands" to remove it from the microwave and leave it on the bench to cool for about 5 minutes, or to about 60°C (just too hot to hold it with your bare hands).
  • Add 1μL of ethidium bromide (10mg/ml) per 100ml of melted agarose and swirl the flask to mix. This will give you a final EtBr concentration of 0.1μg/ml. Dispose of the ethidium bromide tip into the appropriate waste container.
  • At this point you may set-up your gel mold (comb, etc) and pour the gel. Let it sit for 30minutes-1hour for the best looking bands (leaving it for less time can lead to smeary diffuse bands, even if the gel seems firm). Rinse the flask immediately after you pour the gel.
  • Don't forget you need to be using the same running buffer in the gel box as you used for making the gel.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

*Megan N McClean:This protocol could be expanded, and a general "How to pour and run a gel" protocol would be helpful, if anyone has the time to write one up.

*Megan N McClean: Just to repeat: ethidium bromide is bad for you! Wear nitrile gloves, a lab coat, and goggles when working with it. If you spill, let someone know so that we can clean it up.

*Megan N McClean: Small quantities of aqueous solutions containing an ethidium bromide concentration of less than 10 µg/ml (10 ppm) may be flushed down the drain. Non-aqueous solutions and solutions containing an ethidium bromide concentration of greater than 10 µg/ml must be disposed of as hazardous waste. Or use the "tea-bags" available from the Mol Bio stockroom to clean-up liquid waste (the tea bag can then be dried and disposed of as hazardous waste).

References

Contact

or instead, discuss this protocol.


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