Sonication Check

Here are the results from my sonication experiment. This picture shown is the result of running the DNA samples on an agrose gel. Agrose is a type of sugar that polymerizes into a gel. In order to run an agrose gel, you have to load a small amount of DNA into the gel and apply a voltage through the gel. DNA is negatively charged so when the current in applied, DNA will move through the gel toward the positive terminal at the other side of the gel. Inside the gel, DNA pieces of different sizes will move at different rates, similar to how proteins move through a gel in western blots. Larger pieces of DNA move slower than smaller pieces of DNA which allows us to determine what size DNA fragments we have in our sample. 

Let me explain the picture a little bit. The far left and right columns are called DNA ladders. They contain DNA pieces of known size and serve as a marker for how far a specific piece of DNA should run. The bands at the bottom are smaller in size than the bands at the top of the gel. I should also mention that the darker the band, the more DNA there is in that particular location. Going from left to right in the other columns are the DNA samples that I took after I sonicated. Again from left to right, the samples are as follows: 3 cycles, 6 cycles, 9 cycles…up to 24 cycles of sonication. (A cycle of sonication is 30 seconds in the sonicator and one minute on ice). As you can see on the gel, in the samples where I sonicated less, the DNA is larger where as in the samples where I sonicated more, the DNA is in smaller pieces.  The results also show that I should be doing about 15 cycles of sonication to achieve small pieces of DNA.

When I did my first ChAP, I only sonicated for three cycles, which probably wasn’t ideal. However, I’m not sure if the DNA is large enough to cause any significant problems so I’m not sure that the sonication is that big of an issue in my ChAP procedure. This is frustrating because I don’t think that I have a good answer to what went wrong. Since I have the DNA already prepared, I think I’m going to continue with the ChAP protocol to see if the sonication helped to achieve a higher purification. Hopefully it works better this time, but if it doesn’t it means that sonication isn’t the problem that’s causing me to get bad results.