Team:Newcastle/Project/Labwork/MoreProtocols/DNAGel

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<font color="green">'''''Image 1 shows what the agarose solution should look like - as a clear liquid with no solid agarose present'''''</font>  
<font color="green">'''''Image 1 shows what the agarose solution should look like - as a clear liquid with no solid agarose present'''''</font>  
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==Preparing the buffer==
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* The buffer we use for DNA gel electrophoresis is called TAE (Tris-acetate-EDTA) – WE NEVER USE WATER! To make up a stock solution of this buffer at 50x concentration TAE, use:
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# 242 grams of Tris
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# 57.1 ml of Glacial Acetic Acid
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# 100 ml of 0.5M EDTA (pH 8.0)
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* Make up volume to 1 litre using DEIONISED WATER
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* From this stock solution, make up a 1x concentrated TAE solution and to this add 8 microlitres of ethidium bromide.
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Revision as of 15:28, 10 July 2009

Contents

DNA Gel Electrophoresis

Considerations:

- Wear gloves at ALL times
- Keep ethidium bromide in one place and wear gloves when using it. Once you have finished using it OR are walking to other areas of lab, remove gloves and put on a fresh pair. ETHIDIUM BROMIDE IS A CARCINOGEN – DON’T SPREAD IT ALL OVER THE LAB!

Preparing the gel

Image 1: 0.8% Agarose Gel
  • Make up a stock of agarose solution, in which the agarose should constitute 0.8% of the weight/volume (the rest should be made up of the buffer used for the DNA electrophoresis – see step 2 before proceeding)


  • Melt the agarose solution. This should be done in a microwave, initially for 3 minutes on a medium/high setting. After this time has expired take out the solution using gloves and hold up to the light. Give it a swirl and check for any solids within the solution. If it hasn’t completely melted, put it back in the microwave until it does.


***Important note: when putting gel in microwave, keep checking on it regularly (once every 30-40 secs) – it must not be left unattended!***


  • Using gloves, remove the gel from the microwave and check it for any solids. If it has completely melted, leave it somewhere to cool.


Image 1 shows what the agarose solution should look like - as a clear liquid with no solid agarose present

Preparing the buffer

  • The buffer we use for DNA gel electrophoresis is called TAE (Tris-acetate-EDTA) – WE NEVER USE WATER! To make up a stock solution of this buffer at 50x concentration TAE, use:
  1. 242 grams of Tris
  2. 57.1 ml of Glacial Acetic Acid
  3. 100 ml of 0.5M EDTA (pH 8.0)


  • Make up volume to 1 litre using DEIONISED WATER


  • From this stock solution, make up a 1x concentrated TAE solution and to this add 8 microlitres of ethidium bromide.



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