Revision as of 03:30, 11 July 2009

Meetings Calendar

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

Image 2: Bottle of Ethidium Bromide

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:

242 grams of Tris
57.1 ml of Glacial Acetic Acid
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.

Preparing the gel electrophoresis trays

Image 4: Jane showing what we mean by the comb and the DNA gel electrophoresis plate

Image 5: Jane showing the manner in which the comb should be inserted into the plate - the groove near the edge of the plate

Make sure that a clean tray is prepared before carrying out procedure. This must be cleaned using de-ionised water and then with the buffer in which the electrophoresis will be conducted in.

Make sure that the tray has been dried completely using a paper towel.

Make sure that there is a clean, dry comb (cleaned in the same format as the tray).

Insert the comb into the groove of the plate (nearest the end).

Place the tray along with comb in proximity of the electrodes.

20 – 21 June 2009 - Europe workshop (London)
23 – 24 June 2009 - UK iGEM meetup (Edinburgh)
23 October Practice Presentation (Newcastle)
23 October T-shirts are ready
27 October Practice Presentation (Sunderland)
27 October Poster is ready
30 October – 2 November 2009 - Jamboree (Boston)

Newcastle iGEM Twitter
[http://www.facebook.com/home.php#/group.php?gid=131709337641 Newcastle on Facebook]
[http://www.youtube.com/user/newcastle2009igem Newcastle Youtube Channel]

@@ Line 36: / Line 36: @@
 ==Preparing the gel electrophoresis trays==
 [[Image:Team Newcastle iGem 2009 09-07-09 no 2.JPG|200px|thumb|right| Image 4: Jane showing what we mean by the comb and the DNA gel electrophoresis plate]]
+[[Image:Team Newcastle iGem 2009 09-07-09 no 3.JPG|200px|thumb|right| Image 5: Jane showing the manner in which the comb should be inserted into the plate - the groove near the edge of the plate]]
 * Make sure that a clean tray is prepared before carrying out procedure. This must be cleaned using de-ionised water and then with the buffer in which the electrophoresis will be conducted in.
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Team:Newcastle/Project/Labwork/MoreProtocols/DNAGel

From 2009.igem.org