Team:Newcastle/Project/Labwork/MoreProtocols/DNAGel

=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

 * 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.
 * Image 2 shows where the solutions of TAE buffer can be found (the shelf is located near the DNA gel electrophoresis machine, centrifuge and shaker incubator). The top red circle indicates the 50x concentrated TAE buffer; the bottom yellow circle indicates the 1x concentrated TAE buffer. For the electrophoresis, use the 1x TAE buffer. If it runs out, MAKE UP NEW 1x BUFFER FROM 50x TAE BUFFER and place it on the appropriate shelf

Preparing the gel electrophoresis trays

 * 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.
 * Image 3 shows the electrophoresis tray and the comb as separate items - make sure they are both cleaned and dried!
 * Image 4 shows the manner in which the comb should be inserted into the electrophoresis tray - the groove near the edge of the tray should be used. This edge is where the DNA will be placed into the formed wells.

Pouring the buffers and gel

 * Place the buffer into the electrophoresis apparatus first (which should be the 1x TAE). Place lid over tray.


 * When the gel is cool enough but still in liquid form, pour it into a flask (up to 100ml) and to it add 8 microlitres of ethidium bromide. After that, place the 100ml of agarose gel (which should still be in complete liquid form) plus ethidium bromide into the tray, making sure the comb is still in the tray. Make sure that there are no bubbles in the solution – use a pipette end to remove bubbles.


 * After 10 minutes, check to see whether the gel has set (don’t disturb the gel near the comb end – this is where the DNA will be inserted)


 * It may be an idea to get the DNA samples prepared whilst waiting for the gel to set – this can be seen in section 5 – “Preparing the DNA samples”


 * When the gel has set completely, transfer the tray to the electrophoresis apparatus and remove the comb gently – a series of wells should be created. Place lid on tray once completed
 * Image 5 shows what the bottle of ethidium bromide looks like. Use with care and remember to dispose of gloves before walking around lab and after finishing using the ethidium bromide!
 * Image 6 shows the buffer solution + ethidium bromide being added to the tray. The gel should be added afterwards. Notice the comb has been placed in first before the solution is added.
 * Image 7 shows what we mean by the lid of the electrophoresis tray. It should be placed over the electrophoresis machine as soon as the buffers and the gels have been applied.

Preparing the DNA samples



 * Prepare some fresh Eppendorf tubes and to each of them add 9 microlitres of the sample DNA and 1 microlitre of dye.


 * It may be an idea to lightly centrifuge them so that all of the dye and DNA congregates to the bottom to the Eppendorf tube – to do this, place tubes in centrifuge (set for 1 minute) and start the spinning process. Once it reaches maximum speed (13,000 rpm for our centrifuge), stop spinning process.


 * Remove tubes from centrifuge and place in rack in the same order that they are to appear in the lanes of the gel.


 * Make sure that the standard DNA ladder is placed into an Eppendorf tube and placed into the rack before the others. This will be inserted first.
 * Image 8 shows the application of plasmid DNA to the "DNA preparation" Eppendorf tube. Notice that it is a clear liquid, with no precipitate (may need to zoom in to see clearly!). The DNA preparation should be free from protein.
 * Image 9 shows the application of the dye to the "DNA preparation" Eppendorf tube. It appears as a black liquid. Remember, it is a good idea to keep a stock of the dye (the dye in this Eppendorf tube is stock taken from the original conatiner of dye)

Loading the samples into the gel

 * Using a P20 pipette, take up 10 microlitres from the first Eppendorf tube (containing the DNA + dye).


 * Make sure that you can see the row of wells through the water-like solution before doing anything else.


 * With the row of wells in sight, place the pipette end just above the well of choice (for the first entry, try and leave a one well margin). Make sure the pipette is pointed vertically above the well and make sure that the pipette end is not touching the gel or poking into the well.


 * Empty the contents of the pipette (DNA + dye) into the well slowly so that the solution fills the well without dispersing into the solution around the well.


 * Repeat the process for all of the tubes.


 * Once completed, close the lid on the tray and place the electrodes in the appropriate positions. The positive electrode should be located at the end opposite to the comb as DNA is negatively charged and will move towards it.


 * Switch on the power, setting the voltage to 100 volts.


 * Leave for about 30-40 minutes.


 * Image 10 shows Jess pipetting the 10 microlitres of DNA + dye into the wells of the gel. Note that the pipette is pointing just above the well (not into it) and that the pipette is pointing vertically over it. Also note that there is no dispersion around the site of the wells – this is because the solution has been applied slowly!

What you should see

 * Whilst the gel is running, there may be froth visible around the site of the electrodes. This sometimes happens and shouldn’t be cause for concern. If this does happen however, make sure that the temperature isn’t getting to hot


 * Once the electrophoresis has finished, there should be three bands which appear indicating the progress of the reaction. See image 11...


 * Image 11 - this is the gel once electrophoresis has finished. There are three bands visible - these serve as visible markers so that you can track how far the DNA has travelled: a cyan band (nearest the DNA wells), dark blue band and a yellow band (furthest from DNA wells)

What to do next

 * Switch off the electricity and remove the gel from the solution.


 * In a container, take the gel to a dark room and place it under a UV light scanner.


 * Using dials on the scanner, focus and zoom in on the gel to make sure that the resonance and size of the image is good enough for analysis.


 * Either save the image onto the computer, print the image or do both.


 * Image 12 - This is the photograph that should be seen once the gel has been placed in a UV scanner. The DNA standard ladder can be seen in the first lane followed by our experimental DNA in the other lanes (ordered 1-10). Only lane 9's DNA didn't show up in this experiment - this is probably because the E.coli which grew in colony 9 gained resistance to the ampicillin covering the nutrient media by other means.