Fong:DNA gel electrophoresis: Difference between revisions
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==Casting agarose gels== | ==Casting agarose gels== | ||
Choose buffer and agarose gel concentration according to the expected size range of the DNA to be separated (see table). 1% agarose with TAE is a good choice for general procedures. Choose the casting tray and gel comb depending on the number and volumes of the samples to be loaded. The small casting trays need about 50mL total volume, and the large trays about 100mL. | Choose buffer and agarose gel concentration according to the expected size range of the DNA to be separated (see table). 1% agarose with TAE is a good choice for general procedures. Choose the casting tray and gel comb depending on the number and volumes of the samples to be loaded. The small casting trays need about 50mL total volume, and the large trays about 100mL. | ||
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{| class="wikitable" border="1" | |+ '''Final Concentration of Agarose for DNA electrophoresis (%)''' | ||
|+ Final Concentration of Agarose for DNA electrophoresis (%) | |||
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! Size Range (bp) | ! Size Range (bp) | ||
Revision as of 15:12, 7 May 2009
Overview
This page is under construction
See the excellent consensus protocol for an overview. This version is intended to provide instructions specific to the equipment and reagents available in the Fong lab.
Agarose gel electrophoresis is used to separate DNA fragments by size. DNA carries a net negative charge, so in the presence of an electrical current it will tend to migrate towards the positive (red) electrode. Smaller DNA fragments can navigate the agarose gel matrix more easily and therefore migrate further than longer fragments.
Casting agarose gels
Choose buffer and agarose gel concentration according to the expected size range of the DNA to be separated (see table). 1% agarose with TAE is a good choice for general procedures. Choose the casting tray and gel comb depending on the number and volumes of the samples to be loaded. The small casting trays need about 50mL total volume, and the large trays about 100mL.
| Size Range (bp) | 1x TAE | 1x TBE |
|---|---|---|
| 1,000-23,000 | 0.60 | 0.50 |
| 800-10,000 | 0.80 | 0.70 |
| 400-8,000 | 1.00 | 0.85 |
| 300-7,000 | 1.20 | 1.00 |
| 200-4,000 | 1.50 | 1.25 |
| 100-3,000 | 2.00 | 1.75 |
Materials
- 1X TAE or 1X TBE buffer
- Agarose
- Ethidium bromide solution, located in safety cabinet below hood.
Procedure
- Measure appropriate weight of agarose and add to a sterile 500ml flask.
- Add the appropriate volume of buffer, mix by swirling the flask, and microwave for about 1.5-2 min. Use this time to set up the casting tray.
- Allow the mixture to cool enough that you can comfortably touch the flask with your gloved hand, and then add 3.5μL ethidium bromide(for 50mL) and swirl to mix. Dispose of pipette tip in chemical waste.
- Pour mixture into the casting tray, and use the comb to carefully remove any bubbles. Place the comb in the slot and allow the gel to cool to a solid (~5-10min).
- Carefully remove comb and place gel into the gel box so that the samples will run towards the red (+) electrode. Gently pour the same buffer used in the gel over the gel until the top of the gel is just covered.
