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.

(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.