Alm:Agarose gel electrophoresis

From OpenWetWare

Revision as of 14:19, 25 July 2008 by ClarkeS (Talk | contribs)
Jump to: navigation, search

Contents

Overview

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

Materials

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 100s bp fragments of 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 and the gel imager.
  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

NEB Gel Loading Dye, Blue (6X)

is a pre-mixed loading buffer with one tracking dye for agarose and non-denaturing poylacrylamide gel electrophoresis. This solution contains SDS, which often results in sharper bands, as some restriction enzymes are known to remain bound to DNA following cleavage. EDTA is also included to chelate magnesium (up to 10 mM) in enzymatic reactions, thereby stopping the reaction. Bromophenol blue is the standard tracking dye for electrophoresis. It migrates at approximately 300 bp on a standard 1% TBE agarose gel.

1X Gel Loading Dye, Blue (6X):

  • 2.5 % Ficoll 400
  • 11 mM EDTA
  • 3.3 mM Tris-HCl
  • 0.017 % SDS
  • 0.015 % Bromophenol Blue
  • pH 8.0 @ 25°C

IBI (Shelton Scientific) 6x loading dye

15% Ficoll in a special TRIS Dye

Dye #1: Light blue slightly slower than Xylene Cyanol migrating at around 4,oo base pairs in a 1% agarose gel.

Dye #2: Indigo dye migrates similar to Bromophenol Blue at around 600 base pair in a 1% agarose gel.

Dye #3: Magenta dye migrates at around 150 base pairs in a 1% agarose gel.

Contact

  • Arne Materna
  • Sean Clarke

References

Personal tools