USTC/Notebook/Running DNA Gels

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'''[[Team:USTC/Notebook|< Back to Notebook]]'''
'''[[Team:USTC/Notebook|< Back to Notebook]]'''
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== Running DNA Gels ==
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== Agarose Gel Electrophoresis ==
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Preparation
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.  
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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.  
+
*Carefully bring the solution just to the boil to dissolve the agarose, preferably in a microwave oven.  
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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.
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*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.
-
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.  
+
Procedure
-
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.  
+
*After the gel has been prepared, use a micropipette to inject about 2.5 µl of stained DNA.
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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.
+
*Close the lid of the electrophoresis chamber and apply current (typically 100 V for 30 minutes with 15 ml of gel).  
-
Put the gel, together with the rack, into a tank with TAE. Ethidium bromide at the same concentration can be added to the buffer. The gel must be completely covered with TAE, with the slots at the end electrode that will have the negative current.
+
*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

< Back to Notebook

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.
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