Team:EPF-Lausanne/Protocol/Gel

From 2012.igem.org

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Agarose concentration depends on the size of the DNA to be run. We will mostly use 1%.
Agarose concentration depends on the size of the DNA to be run. We will mostly use 1%.
VOL is the desired volume of gel in ml:
VOL is the desired volume of gel in ml:
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[[File:Team-EPF-Lausanne-Protocols-1kb-ladder.gif‎|thumb|1kb ladder bands]]
# Add 0.01*VOL g of agarose to a clean glass bottle.
# Add 0.01*VOL g of agarose to a clean glass bottle.
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# Wait until cold and solidified.
# Wait until cold and solidified.
# Carefully remove comb.
# Carefully remove comb.
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# Place the box in the voltage generator.
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# Place the box in the electrophoresis chamber.
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# Cover the gel with buffer. (What buffer?)
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# Fill up the electrophresis chamber with 1x TAE buffer.
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# Add blue dye to the DNA samples and ladder marker. (Which dye? How much?)
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# Add blue dye to the DNA samples and ladder marker (6x loading buffer, that is 10 µl in 50 µl of DNA solution).
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# Inject 20 µl of ladder marker in the first well.
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# Inject 30 µl of ladder marker in the first well (that's 1 µg of DNA).
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# Inject 20 µl of each DNA solution in the other wells.
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# Inject 60 µl of each DNA solution in the other wells.
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# Ready to run.
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# Set voltage to 70-90 V and run for 30-40 min (DNA travels from - to +).
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[[File:Team-EPF-Lausanne-Protocols-1kb-ladder.gif‎|frameless]]
 
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Revision as of 14:23, 4 July 2012

Protocol: Gel electrophoresis


Agarose concentration depends on the size of the DNA to be run. We will mostly use 1%. VOL is the desired volume of gel in ml:


  1. Add 0.01*VOL g of agarose to a clean glass bottle.
  2. Pour VOL/50 ml of 50xTAE in a graduated cylinder. Fill up to VOL ml with di water.
  3. Add the resulting VOL ml of 1xTAE to the glass bottle with agarose.
  4. Microwave, at 7, the bottle (no cap!) until it boils.
  5. Carefully remove bottle (can be super heated!) and check for the total absence of particles. Microwave again if needed.
  6. Prepare a gel box, with comb, and fill it up with the agarose solution (maybe not the whole solution is needed).
  7. Add 0.05 µl per ml of gel in the box of Red Gel (it's in the iGEM drawer) and stirr until disolved.
  8. Wait until cold and solidified.
  9. Carefully remove comb.
  10. Place the box in the electrophoresis chamber.
  11. Fill up the electrophresis chamber with 1x TAE buffer.
  12. Add blue dye to the DNA samples and ladder marker (6x loading buffer, that is 10 µl in 50 µl of DNA solution).
  13. Inject 30 µl of ladder marker in the first well (that's 1 µg of DNA).
  14. Inject 60 µl of each DNA solution in the other wells.
  15. Set voltage to 70-90 V and run for 30-40 min (DNA travels from - to +).

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