Richard Lab:Agarose Gel Electrophoresis

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<center>This protocol is for the typical separation and purification of DNA done in the Richard Lab.   
<center>This protocol is for the typical separation and purification of DNA done in the Richard Lab.   
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The consensus [[agarose gel electrophoresis | electrophoresis protocol]] should be consulted if deviating from the procedure outlined here.</center>
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<br>The consensus [[agarose gel electrophoresis | electrophoresis protocol]] should be consulted if deviating from the procedure outlined here.</br></center>
===Procedure===
===Procedure===

Revision as of 14:39, 12 June 2009

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This protocol is for the typical separation and purification of DNA done in the Richard Lab.
The consensus electrophoresis protocol should be consulted if deviating from the procedure outlined here.</br>

Procedure

  1. Place the gel tray perpendicular in the electrophoresis to create a casting bay.
  2. Wipe the tray clean with a Kim-wipe and level the tray using the bubble level.
  3. Weigh out the desired amount of agarose and add it to 100ml of 1X TBE in a 300ml flask.
  4. Microwave the flask on high for 80 seconds. Be sure that it does not boil over.
  5. Swirl the microwaved agarose in the flask until the solution becomes clear.
  6. Pour the melted solution into the casting bay and insert the comb.
  7. Prepare the DNA ladder by combining the following:
    1. 10ul DNA ladder
    2. 1ul SYBR green (100X)
    3. 1ul Bromophenol Blue
  8. Prepare the DNA Samples by combining the following:
    1. 40ul DNA preparation
    2. 4ul SYBR Green (100X)
    3. 4ul Bromophenol Blue
  9. Remove the comb from the cured gel and realign the gel in the chamber.
  10. Adjust the buffer level by adding 1X TBE to the chamber until the buffer just covers the gel. The buffer can be reused a few times.
  11. Pipette the samples into the wells
  12. Apply 150 volts and run for approximately 60 minutes.
  13. Photograph the gel using the UVP transilluminator system.

The amount of agarose to use in your gel depends on the DNA in question. As a rough guide: Agarose (g/100mL) 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

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