Team:Washington/Protocols/gel electrophoresis

From 2011.igem.org

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# Measure out the appropriate mass of agarose into a beaker with the appropriate volume of buffer (see the documentation for your gelbox -- 50mL makes a good, thick gel for a 7x10cm gelbox).   
# Measure out the appropriate mass of agarose into a beaker with the appropriate volume of buffer (see the documentation for your gelbox -- 50mL makes a good, thick gel for a 7x10cm gelbox).   
# Microwave until the agarose is fully melted.  This depends strongly on your microwave, but a 90 seconds at full power or 3 minutes at half power seem to provide decent results.  As long as you do not burn the agarose and nothing bubbles over, this step is robust.
# Microwave until the agarose is fully melted.  This depends strongly on your microwave, but a 90 seconds at full power or 3 minutes at half power seem to provide decent results.  As long as you do not burn the agarose and nothing bubbles over, this step is robust.
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# Let the agarose cool on your bench until touching the bottom of the beaker with your bare hand doesn't burn you (~5 minutes for a 50mL gel).  At this point add your DNA stain, e.g., ethidium bromide.  The beaker will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles.
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# '''From here on, a heat protective glove should be used any time the heated flask must be touched!'''
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# Pour the agarose solution into the gelbox.  Carefuly pop or shove to the side any bubbles, put in the comb, and let it cool for about 30 minutes, until the gel is solid.
+
# Let the agarose cool on the bench for ~5 minutes.   
 +
# At this point add your DNA visualization reagent, e.g., ethidium bromide at a volume appropriate for the amount of melted agarose.  This amount will depend on the concentration of the stock solution of the stain. 
 +
# The flask will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles, and should therefore be periodically swirled while the agarose is cooling.
 +
# Pour the agarose solution into the gel casting apparatus.  A pipette tip should be used to pop or shove to the side any bubbles.
 +
# After bubbles have been popped, the comb should be inserted, and the gel should be allowed to cool for about 30 minutes, until solid.
 +
# Because removal of the comb too early can cause the wells to collapse on themselves, one should always poor a gel prior to prepping the samples.
Adapted thanks to[http://openwetware.org/wiki/Agarose_gel_electrophoresis OpenWetWare protocols].
Adapted thanks to[http://openwetware.org/wiki/Agarose_gel_electrophoresis OpenWetWare protocols].

Revision as of 02:26, 16 September 2011


Agarose Gel Electrophoresis

General Procedure

  1. Cast a gel
  2. Place it in gel box in running buffer
  3. Load samples
  4. Run the gel
  5. Image the gel


Casting Gels

The amount of agarose to use in your gel depends on the DNA in question. Use the following table as a rough guide:

Agarose Concentration (g/100mL) Optimal DNA Resolution (kb)
0.5 1 - 30
0.7 0.8 - 12
1.0 0.5 - 10
1.2 0.4 - 7
1.5 0.2 - 3
  1. Measure out the appropriate mass of agarose into a beaker with the appropriate volume of buffer (see the documentation for your gelbox -- 50mL makes a good, thick gel for a 7x10cm gelbox).
  2. Microwave until the agarose is fully melted. This depends strongly on your microwave, but a 90 seconds at full power or 3 minutes at half power seem to provide decent results. As long as you do not burn the agarose and nothing bubbles over, this step is robust.
  3. From here on, a heat protective glove should be used any time the heated flask must be touched!
  4. Let the agarose cool on the bench for ~5 minutes.
  5. At this point add your DNA visualization reagent, e.g., ethidium bromide at a volume appropriate for the amount of melted agarose. This amount will depend on the concentration of the stock solution of the stain.
  6. The flask will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles, and should therefore be periodically swirled while the agarose is cooling.
  7. Pour the agarose solution into the gel casting apparatus. A pipette tip should be used to pop or shove to the side any bubbles.
  8. After bubbles have been popped, the comb should be inserted, and the gel should be allowed to cool for about 30 minutes, until solid.
  9. Because removal of the comb too early can cause the wells to collapse on themselves, one should always poor a gel prior to prepping the samples.

Adapted thanks to[http://openwetware.org/wiki/Agarose_gel_electrophoresis OpenWetWare protocols].


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