Back At It

This week I returned to working with my plant samples.

At the end of last semester I began attempting DNA extractions on the samples of the Foothills Palo Verde using the Wizard extraction kit. Unfortunately there were no usable results from this. The protocol for this kit required the leaf sample to be ground up in solution and then pushed through a min-icolumn (like a filter) with a 5mL syringe. No matter how I ground the sample or changed the dilution rate, I had to exert a very large amount of force to push the sample through the media. Every time I attempted this, I essentially compressed the 4.5mL volume of air down to approximately 1mL before anything would pass through the column, and then it all went! I assumed that this was basically bypassing the filtration that was in place (blowing it out).

This semester I started extracting using a system from Sigma-Aldrich that was an entirely different approach. This system did not require the grinding of the leaf material. The "quick and dirty" extraction method (this is how Matt and Josh referred to it) relied upon heat and an extraction solution. The end results were much the same. When gel electrophoresis was done, there was no traveling of DNA through the gel. Maybe this was because the extraction method was in fact a little too quick and dirty?

This week I decided to try to combine the two methods. I used a sample from the Sigma-Aldrich sample and then passed it through the wizard kit. My thinking was the Wizard kit didn't seem to handle the material although it should have yielded a better end product, and the SA system was able to handle the material but was a dirty extraction, why not try to use the better aspects of each and clean the dirty extraction with the finer system. Sounds logical right?

Well... it didn't work.

Again there were no visible bands in the gel. However it is of note that the genetic ladder or mass ruler that I used did not appear to run correctly either. The ladder is used as a comparison method for DNA. In a nutshell, gel electrophoresis works based on the size of DNA fragments. The samples are loaded in the gel and it is placed into the gel box that is filled with a liquid buffer (TAE). The gel is oriented so the DNA (which has a negative charge) is located near the negative pole of the electrophoresis system and will be drawn through the gel to the positive end when 120 volts is applied to the system (for 30 - 45 minutes). The larger the piece of DNA, the slower it will travel through the gel. A properly run sample will show different bands separated through the gel by the distance it travels. The ladder should have had clear solid bands at different distances through the gel. This was not the case, it was basically a long smear as shown in the picture below.

First off, yes the top of the gel is melted. It was run longer than normal and that is the result. from left to right there are 8 "wells" for samples. The first two were my DNA extraction,then an empty well, then two of one ladder, another opening, and then two of another ladder. This image is under UV light, hence the glow. The smear I mentioned is obvious. There should be separate distinct bands at varying lengths down the gel. I am not sure if there is actually DNA in the wells of my sample or not. There is a discernible fluorescence in the well but that may just be the dye, I am not certain.

What I do know is, there is something wrong in either my gel protocol or methods. I hate to admit it, but it is most likely user error...

The gel is composed of 1% agarose to buffer solution. The buffer solution is a dilution of 50x TAE. Have I erred in my formulations of these two products? Maybe I need to use a different concentration of one or both of these products? Either way, I want to resolve this as quickly as I can. I would certainly like to move on with the rest of this project, I a m really excited to hopefully be able to prove the Palo does do some reproduction through cloning.