Protocol for Agarose Gel Preparation
- Weigh out the required amount (depending on the gel percentage) of agarose into an Erlenmeyer flask.
- Add the appropriate volume of either 1X TBE or 1X TAE buffer and swirl to mix.
- Weigh the flask with the solution.
For high percentage gels (3-5%): add an excess amount of distilled water to increase the weight by 10-20%.- Boil the mixture in a microwave oven (at middle power) until the agarose melts completely; swirl the flask several times while boiling. To prepare the highest quality agarose gels of any percentage,an additional 3-5 min of boiling after completely melting the agarose is recommended. A significant amount of water evaporates during this procedure and therefore restoring of the initial weight (in step 5) is required to obtain the desired percentage gel.
- Weigh the flask again and if necessary, add hot distilled water to restore the initial weight.
For high percentage gels (3-5%): check (by weighing) that the excess 10-20% of water has evaporated and, if needed, continue boiling to remove the said excess, or add hot distilled water to restore the initial weight.- Optional: for an intensified gel staining add ethidium bromide to a final concentration of 0.5 µg/ml. Then, mix well and heat the mixture for an additional minute without boiling.
- Cool the solution to 65-70°C. Pour it carefully on a clean casting plate with an appropriate comb. If bubbles appear, push them carefully away to the sides with a tip.
- Solidify the gel for about 30 min before using. Low percentage LM agarose gels can be solidified at +4°C.
- Immerse the gel into the desired electrophoresis buffer and load samples.
- After electrophoresis the gel can be stained with ethidium bromide, SYBR® Green I or by any other staining technique.
Warning
Hot agarose solution should be handled very carefully.
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Updated gegužės 09, 2007 12:57
