Gel Electrophoresis

     A gel electrophoresis allows a scientist to analyse DNA. Current is passed through a gel (from - to +), and DNA molecules migrate to the + pole. DNA molecules of different lengths separate, and the shortest segment will move the farthest along the gel. Say we have a plasmid that is cut at one site. This results in a linear DNA fragment, of the same size as the original DNA. Now suppose that we cut it twice. There are now two linear fragments, and one is probably bigger than the other. These two will separate out and will show up as two different bands on the gel. The shorter one will be farther down because it can travel faster.
     If you completed an analysis on the CDS of the plasmid you have digested, you can create a virtual gel that you can later use to see if your RE Digest worked. Go to http://tools.neb.com/NEBcutter2/, and enter your CDS (filter it first using http://www.bioinformatics.org/sms2/filter_dna.html). Then choose circular and hit submit. From there, click on "custom digest" and enter the enzymes you used (in this case, EcoRI and PvuII; your CDS should be the CDS for PGBR22). Then you can click on "view gel" (enter 1% agarose and 1kb DNA marker). If you do this for EcoRI, PvuII, and EcoRI & PvuII, you can see the relative band separations. This is what your actual gel should look like.

To make your gel:

Add 0.7g agarose to 70mL TAE buffer (this will give you 1% agarose). Microwave the solution until it is boiling. Remove the flask from the microwave using insulated gloves and wait for it to cool down, swirling the mixture occasionally. When it is cool enough (you should be able to hold it for 10 seconds), add 0.8uL of ethidium bromide (this is a carcinogen, so be careful!) and swirl it until it is clear. Then, pour the solution in to the casting tray and place the comb (you only need one for an RE digest) in the solution. Wait for the gel to solidify.





To run your gel:

Carefully remove the casting tray and take out the comb. You should be able to see the wells if you look at the gel from a bird's eye view. Place this in the gel electorphoresis apparatus (pictured at the beginning of this post) and fill the chamber up to the fill line with TAE buffer. Then, connect the wires to the corresponding colours on the machine, and set at 105V. If you set the voltage higher than this, the DNA will run faster, but the gel may be prone to breakage (the high voltage makes the solid less stable, and cracks can easily form).

To check your gel:

Carefully remove the gel block from the casting tray, taking caution not to break or crack the gel (this will make viewing it harder), and place it in the UV viewing machine. Flick the switch for the UV light to high, turn it on, and then switch it to low. DO NOT LOOK AT THE UV LIGHT! Find the viewer on the desktop of the computer you are at, and click on it. If you look through the top of the viewing machine, you can see how far your gel has progressed. Use the viewer on the computer to capture a screen shot for future reference. An example is shown below:

However, this does not match the virtual example from the CDS analysis. This suggests that there may be some contamination in the stock solution of the restriction enzymes, or that some of the DNA may have floated from one well to another.