(about) TIme for Progress.

The reagents for the extraction buffer have arrived and I will be able to run the wizard kit again early next week. It is my hope that it will be more successful this time around.

While waiting for the reagents I went ahead and conducted another extraction with the Sigma-Aldrich kit. The genomic DNA results were FAR better than they had been previously! However, there may still be an issue. The DNA in the gel travelled much further than genomic DNA typically does, and the ladder is still coming out strange. The only causes I can think of that may be in effect here are either the ratio of agarose to TAE buffer in the gel is off, or the dilution of the TAE itself is off. I have checked, rechecked, and checked again my math on these products. I feel like I am really reaching to find a problem here.

This is the gel as viewed under uv light. The faint dotted horizontal line represents the wells that the samples were placed in. the purple band on the left is the extracted genomic DNA. Notice how far it has travelled in the gel. It is my understanding that it should have traveled less than half that distance. Genomic DNA is large and should not travel so readily through the gel. The vertical streak on the right is the ladder or molecular marker that I used. It is also strange in that it should have not been a amear like this.

A closer image of the same gel.

I am interested to see what will come from running a PCR amplification on this extraction sample. I will run the PCR once I have extracted DNA through the wizard kit next week and run both samples at the same time.

On another note, I will be heading out to the sample plot at Squaw Peak with Matt Haberkorn tomorrow afternoon to record data for the 3d plot map I am creating. I will use this data coupled with some root mapping to complement my genetic research into cloning in the Palo Verde. We will also be doing some foliage cover estimates to compare and contrast the growth habits of the Palo in the washes as opposed to in the uplands. There may even be some soil sampling in the works to round out the data as a whole. Looking forward to be out on the site. It should be a nice way to wrap up a rather busy week.

again, and again, and again, again...

Again, I will be starting over... I feel as though I have said that before. Early on this week I made the decision to retry the higher quality extraction with the wizard kit I used at the end of last semester. There was one slight hitch. The extraction buffer I used is no longer viable. Embarrassingly enough,  as I recall, the buffer I used last semester was just given to me, and I had no idea of its composition or who made it. Much of this week was then spent researching different extraction methods and buffers that would work for me. MANY of these call for the leaf sample to be frozen in liquid nitrogen and then ground up before adding buffers - not really an option for me. Also of note many methods require the use of phenol-chloroform (two nasty chemicals). I wasn't told not to use phenol-chloroform, but it felt as if it were implied that another method may be better.

After reviewing a rather large amount of scientific literature (predominately journals), I did find a method that didn't require the above. Unfortunately not all of the reagents required for the buffer were on hand, but we anticipate delivery early next week. The components of the buffer are:

200 mM Tris HCl pH 7.5 This maintains a stable pH in solution as cells are lysed.

250 mM NaCl Provides Na+ Ions that bond to the negatively charged phosphate groups that are part of DNA (if the Na+ didn't do this the molecules would repel each other)

25 mM EDTA Is a chelating agent that neutralizes metal ions like Mg2+ and CA2+ which are needed by DNA polymerase and other enzymes. Doing so essentially stops the action of these enzymes and preserves the DNA in it's intact state.

0.5% SDS is a detergent.

 

I can't wait to finally get some results! The end of the semester is approaching much faster than I care to admit! 

For general information good results would look something like this:

Image from Promega's website (the maker of the wizard kit.

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.