USTC/Notebook/Agarose Gel Electrophoresis

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Agarose Gel Electrophoresis
Materials


 * DNA, the thing you want to analyze/cut out. Measure concentration in nanodrop beforehand.
 * 1xTAE buffer.
 * Gel-red dye, 10,000x.
 * DNA Marker DL2000 and DL15000.
 * Melted Agarose in 1xTAE.
 * melting appropriate amount of Agarose mixed in TAE in the microwave. (eg. to prepare 100ml of melted 1% agarose, mix 1g of agarose in ~99ml 1xTAE in a foil-covered flask and microwave for 4+min; keep an eye on it every couple of minutes when you microwave so it doesn't boil over; be careful to wear gloves when handling hot liquid containers). Can leave on bench and re-melt every time you need it.

Preparation


 * Make a 1% agarose solution in 100ml TAE, for typical DNA fragments. A solution of up to 2-4% can be used if you analyze small DNA molecules, and for large molecules, a solution as low as 0.7% can be used.
 * Carefully bring the solution just to the boil to dissolve the agarose, preferably in a microwave oven.
 * Let the solution cool down to about 60 °C at room temperature, or water bath. Stir or swirl the solution while cooling, Wearing gloves from here on.
 * Add 10 µl Gel-Red(10000X) per 100 ml gel solution for a final concentration of 0.5 ug/ml. Be very careful when handling the concentrated stock.
 * Stir the solution to disperse the gel-red, then pour it into the gel rack.
 * Insert the comb at one side of the gel, about 5-10 mm from the end of the gel.
 * When the gel has cooled down and become solid, carefully remove the comb. The holes that remain in the gel are the wells or slots.
 * Put the gel, together with the rack, into a tank with TAE. The gel must be completely covered with TAE, with the slots at the end electrode that will have the negative current.

Procedure


 * After the gel has been prepared, use a micropipette to inject about 2.5 µl of stained DNA.
 * Close the lid of the electrophoresis chamber and apply current (typically 100 V for 30 minutes with 15 ml of gel).
 * The colored dye in the DNA ladder and DNA samples acts as a "front wave" that runs faster than the DNA itself. When the "front wave" approaches the end of the gel, the current is stopped.
 * The DNA is stained with gel-red, and is then visible under ultraviolet light.