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USTC/Notebook/Running DNA Gels
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| + | == Agarose Gel Electrophoresis == | ||
| - | + | Materials | |
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| - | + | *'''DNA''', the thing you want to analyze/cut out. Measure concentration in nanodrop beforehand. | |
| - | + | *'''1x'''TAE 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 | |
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| - | + | *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. | ||
Latest revision as of 15:34, 28 October 2008
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.
