Alm:Agarose gel electrophoresis

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Current revision (12:02, 15 January 2009) (view source)
(IBI (Shelton Scientific) 6x loading dye)
 
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==Materials==
==Materials==
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*6X loading dye
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*[[Agarose_gel_loading_dye|6X loading dye]]
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*[[TAE]] agarose 1%
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*[[TAE]] + agarose 1%
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*1X TAE (~200ml)
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*1X [[TAE]] (~200ml)
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*gel box (casting tray optional)
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*gel box (casting tray optional) and power supply
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*power supply
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*[[Ethidium Bromide]] staining solution
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*[[Ethidium bromide]] staining solution
+
*used TAE destaining solution
*used TAE destaining solution
*UV box/gel imager
*UV box/gel imager
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#Add 6x loading dye to your samples.   
#Add 6x loading dye to your samples.   
#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
#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
-
#Set the time and settings on the power supply by pressing Mode to change fields. (typical settings for DNA: 90V constant voltage, 45 minutes)
+
#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)
#Slide on the lid and start the power supply.
#Slide on the lid and start the power supply.
===Staining and visualization===
===Staining and visualization===
-
 
+
*'''Wear blue/purple nitrile gloves when handling the items at the staining station and the gel imager.'''
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*'''Wear blue/purple nitrile gloves when handling the items at the staining station.'''
+
#Place the gel in the plastic box in the hood.
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#Place the gel in the plastic box.
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#Stain for 2 minutes to 1 hour in EtBr solution (depending on the concentration).  Cover the plastic box with the cardboard lid to prevent bleaching of the dye.
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#Stain for 1 hour in EtBr solution (concentration??).
+
#Pour the EtBr back into the light-proof bottle covered in aluminum foil.
-
#Pour the EtBr back into the light-proof bottle.
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#Destain with used TAE for 30 seconds to 2 minutes, rocking the box gently.
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#Destain with used TAE for 2 minutes.
+
#Pour the TAE back into the bottle with the funnel.
-
#Pour the TAE back into the bottle.
+
#Image the gel on a UV light box or the computerized gel documentation (geldoc) system in the Thompson lab or Polz lab.
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#Image the gel on a UV light box or the computerized gel imager in the Polz lab.
+
#Print out the image for your notebook, annotating the lanes, marker, and bands.  Also note electrophoresis details like amount of DNA loaded, dye, voltage, time.
-
#Print out the image for your notebook, annotating the lanes, marker, and bands.
+
#Note agreement with/variation from expected result.
#Note agreement with/variation from expected result.
==Notes==
==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
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*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 4,000 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==
==Contact==
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==References==
==References==
<!-- If this protocol has papers or books associated with it, list those references here.  See the [[OpenWetWare:Biblio]] page for more information. -->
<!-- If this protocol has papers or books associated with it, list those references here.  See the [[OpenWetWare:Biblio]] page for more information. -->
-
<biblio>
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*[[Agarose_gel_electrophoresis]]
-
</biblio>
+
[[Category:Protocol]]
[[Category:Protocol]]

Current revision

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 in the hood.
  2. Stain for 2 minutes to 1 hour in EtBr solution (depending on the concentration). Cover the plastic box with the cardboard lid to prevent bleaching of the dye.
  3. Pour the EtBr back into the light-proof bottle covered in aluminum foil.
  4. Destain with used TAE for 30 seconds to 2 minutes, rocking the box gently.
  5. Pour the TAE back into the bottle with the funnel.
  6. Image the gel on a UV light box or the computerized gel documentation (geldoc) system in the Thompson lab or Polz lab.
  7. Print out the image for your notebook, annotating the lanes, marker, and bands. Also note electrophoresis details like amount of DNA loaded, dye, voltage, time.
  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 4,000 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

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