Alm:Agarose gel electrophoresis

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Materials)
Line 5: Line 5:
*6X loading dye
*6X loading dye
-
*[[TAE]] agarose 1%
+
*[[TAE]] + agarose 1%
-
*1X TAE (~200ml)
+
*1X [[TAE]] (~200ml)
-
*gel box (casting tray optional)
+
*gel box (casting tray optional) and power supply
-
*power supply
+
*[[Ethidium Bromide]] staining solution
-
*[[Ethidium bromide]] staining solution
+
*used TAE destaining solution
*used TAE destaining solution
*UV box/gel imager
*UV box/gel imager

Revision as of 15:49, 20 September 2007

Contents

Overview

How to use gel electrophoresis to separate, measure and visualize DNA pieces.

Materials

  • 6X loading dye
  • TAE + agarose 1%
  • 1X TAE (~200ml)
  • gel box (casting tray optional) and power supply
  • Ethidium Bromide staining solution
  • used TAE destaining solution
  • UV box/gel imager

Procedure

Electrophoresis

  1. Microwave TAE agarose 1% to melt. Pour 30 ml for a small gel into a small flask/beaker to cool a bit, then pour into the gel box with the correct comb for your size/number of samples.
  2. Add 6x loading dye to your samples.
  3. Make sure the red electrode is at the opposite end from the wells. You can check the progress of the DNA with the loading dyes to check the direc
  4. Set the time and settings on the power supply by pressing Mode to change fields. (typical settings for DNA: 90V constant voltage, 45 minutes)
  5. Slide on the lid and start the power supply.

Staining and visualization

  • Wear blue/purple nitrile gloves when handling the items at the staining station.
  1. Place the gel in the plastic box.
  2. Stain for 1 hour in EtBr solution (concentration??).
  3. Pour the EtBr back into the light-proof bottle.
  4. Destain with used TAE for 2 minutes.
  5. Pour the TAE back into the bottle.
  6. Image the gel on a UV light box or the computerized gel imager in the Polz lab.
  7. Print out the image for your notebook, annotating the lanes, marker, and bands.
  8. Note agreement with/variation from expected result.

Notes

Contact

  • Arne Materna
  • Sean Clarke

References

  1. []
Personal tools