Wednesday, February 10, 2010

Gel purification



One of our immediate aims these days is to construct a plasmid that will allow us to create "promoter reporters". This involves steps of DNA cloning, which is like building a puzzle from given pieces of DNA.

Roughly speaking cloning is based on three types of steps:

  • PCR amplification - in which we amplify a region of DNA by using primers.
  • Restriction - where we cut a piece of DNA by an enzyme that cut at a specific place and leaves a specific single strand end that can be then used for puzzle building.
  • Ligation - combining pieces from restriction reactions and letting them combine into complete DNA pieces. 
One of the problems we need to face is that after such steps we don't have complete effect. Some by products may be left in the resulting tube. Thus, we want to purify just the right product out.

One technique to do that is to run the product on a gel. This separates the products based on their length - smaller pieces of DNA "run" faster on the gel and will be at lower points.

 

On the left we see a run of premixed "ladder" of bands. The two bright ones are at 1000bp and 5000bp. The next three columns (or lanes) are three samples. The first one has a nice clean product. The second has a mix of a clean product and a "smear" that looks like the negative control on the third lane.

The picture you see is fluerscence of a DNA-binding dye in response to UV light. When we look at the gel in daylight we only see three dyes at 10, 500, and 5000bp length.


To purify, we cut out the piece of the gel. To do so, we need to examine it on a UV-table. This table emits UV light and then we can see the fluorescent bands by eye.




The problem is how to work so not to get too much of UV radiation on yourself. Moreover, the UV exposure can damage the DNA. Thus, we want to work quickly. Using a scalpel, we cut the bands and put them in tubes for later processing.



1 comment:

Moran said...

Good thing you have your ski goggles at reach :)