Good news of a sort

Last Friday I ran gel electrophoresis with a 0.5% agarose gel instead of the normal 1% gel. I did not get the results I had hoped for, but I did observe some travel in the gel!

In lane number 5 (from left), a very faint band can be seen about halfway down. I consulted with Matt and Josh, the leading thought is that something is working (the band though faint is still there), but not working well. It is believed the specific primers used are not effective. This is not a very big issue at all. There are seven different UPRs (Universal Rice Primers) currently available in the lab, four of which I have yet to use. Hopefully one of the other four will be able to amplify an SSR (Simple Sequence Repeats) in my Palo samples.

Now I need to conduct another DNA extraction from leaf samples, check it with gel electrophoresis, run a PCR with each of the four URPs, and another gel with each of the PCR results, analyze the results to see which, if any, is successful, and then apply the whole process to the each of the samples I collected... Oh wait... what's that? The semester is over next week?

given that, I have had to adapt my poster presentation and research paper to focus on the observable data collected. Hopefully next semester my participation in this program will be renewed and I will be able to continue with this research.

Here we go again.

It seems that every week as I finish writing my blog update I think, maybe next week will be the week when I will be able to post positive results.

Not the case.

Don't get me wrong, I am not discouraged in the least. If anything, the lack of results is a motivator. Getting good genetic evidence of what I am trying to prove has now become sort of a personal challenge (or vendetta?). I am running out of time for this semester though.

Upon completion of the PCR amplification of the DNA extraction, I conducted another gel electrophoresis. As I have seen many times this semester, the material did not travel in the gel. Under UV light, the wells containing the PCR results from the Sigma extraction did glow, but there was no evident travel through the gel.

Electrophoresis separates the molecules based on size. Larger DNA strands will travel much slower than the smaller ones. After a period of time with 100 volts pulling the negatively charge DNA molecules toward the negative pole of the gel box, a separation of the fragments can be seen. Based on this principle behind the separation, I am now questioning if the issue has to do with the size of my sample's DNA fragments. Is it possible that the fragments are simply too big to travel through the gel? In my research I have not come upon anything regarding this.

The gel used is a 1% agarose gel. It is made by adding 1 gram of molecular grade agarose to 100mL of 50x TAE buffer, and heating/mixing it to a clear solution and then pouring it into a mold (the gel box tray) and allowing it to cool. My newly developing theory is that if the DNA fragment is too large to travel through the gel, why not alter the concentration of agarose to make it easier for the fragment to travel. I have no idea if it will work, but today I made a 0.5% agarose solution and poured a gel. I did not have enough time to run the gel, but I will be able to do so tomorrow. Maybe it will work, maybe not.

I am now up against the wall as far as being able to present results at MetroTech, and complete my research paper. Should I obtain the positive results I hope for tomorrow, I am not sure that I have enough time left to interpret them. I believe what I am going to have to do is focus on structuring poster for presentation and the my research paper around the observational work I have done - mapping and imaging of the site it itself showing the spatial interrelation of the subjects and their respective root systems (or better stated the portions of the root systems that can be seen). This will not be near as exciting as what I hoped to accomplish, but it is something. I do not know if I will be selected for renewal of the S-STEM scholarship for a third semester, but I certainly hope so. I want to prove the Palo Verde does reproduce through cloning in addition to sexual reproduction.

This is the best picture of potential cloning in Parkinsonia microphylla I have taken. It is quite obvious that the root system coming from the main portion of the biomass on the left is in fact sending up additional shoots. Notice the large developed shoot on the right and the smaller/newer shoots between the two. They all originate from the same root system.

I think we may have a winner

On Monday I completed an extraction with the new method I devised and ran a gel with it as well as the results from the previous Sigma Aldrich extraction. My new extraction did not seem to produce any results, but the Sigma extraction again showed a nice heavy band as seen below.

The wells from left to right: 1 - the new extraction method, 2 - the Sigma extraction, 3 + 4 - empty, 5 - ladder, 6 - empty, 7+8 - samples Josh asked me to run (Staphylococcus aureus - I don't know the extraction method). 

It certainly is nice to see a big heavy band of genomic DNA. I had intended to conduct a PCR this week using the Sigma extraction but my plans changed... Tuesday I was unable to be in the lab as normal due to a honey bee swarm taking up residence in my house! This required me to bee there (nice pun?) Tuesday. I was made aware that the rough draft of our research paper for this semesters projects is due Thursday (today). For some unknown reason I was under the impression that I had another week. So, I spent a fair amount of time yesterday in the lab working on the draft, and more than a few hours working on it late last night. There is a downside to working late into the night - sometimes mistakes are made, sometimes they are big ones, sometimes you evidently don't save the work you are doing... I am not all the way back to square one, but I lost a lot of work. 

I decided to clear my head and get into typing mode by posting this edition of my blog. Now it's time to get back on the paper. 

and awaaaaaay we go...

Denied...

This week I finally heard back from the Estrella Mountain Community College Student Conference. They regret to inform me that they have not accepted my research proposal for this years conference. This does not worry me as I will find another outlet to present my data. The notification E-mail from EMCC contained a couple of abbreviated comments on my proposal that I find interesting.

1: "Seems to be two projects here..., reproduction and adaptation to washes... It is a bit muddled."

I feel that my proposal pretty clearly stated that the reproduction (cloning) IS an adaption to thriving in the washes.

2: "Not sure if his methodology will actually yield the results he is looking for."

I am recording qualitative (observable) data that shows cloning in the Palo Verde, and supporting that, I am conducting genetic analyses to prove that cloning is occurring. Just exactly what other methodology could be used to do this?

OK complain fest is over!

Matt Haberkorn and I went back to the sample plot at Squaw Peak and recorded topographical data from the terrain in a 3 coordinate system. Using this data I was able to create this site map:

This data was collected by the use of two meter sticks with levels attached. The first was held vertically (plumb) at the "0" mark of the transect line and the second was extended a full meter from the first and lowered until the far end connected with the bank. When held level, this indicated the elevation change to the far point. The vertical stick was then moved up the bank to the exact spot the horizontal stick made contact and the process repeated until we reached a point beyond the highest specimen that I collected leaf samples from. We then reset and began from the one meter mark on the transect line, and continued until the entire plot was recorded on a one meter grid.

Matt and I at work

We will be returning tomorrow to collect data and high resolution images of root systems. We decided that we have a bit better of a method to plot the actual location of the specimens.