Que sera sera

This week, I reviewed my latest protocol in order to locate all of the chemicals required, most of which were already on hand in the lab. There were only two things on my "shopping list" that needed to be ordered: sarkosyl and proteinase k.

Sarkosyl is a surfactant (read detergent). Surfactants work on the basis of lowering the surface tension of liquids they are introduced to. For example oil and water do not "mix," with the introduction of the proper surfactant they could be mixed. Sarkosyl's role in DNA extraction is in the disruption of membranes that is, it renders the proteins within the cell membrane soluble, thereby assisting in lysing the cell.

Proteinase k is an enzyme (hence the "ase" part of its name) that acts upon proteins (the protein part of its name). It will essentially digest proteins that contaminate the extraction results. It will also act upon any nucleases (that are present naturally) and negate their breaking of nucleotide chains (DNA strands).

So the order was made and I'm just waiting on these two items to come in and then I can create my reagents and off I go.

or so I thought...

Upon detailed examination of my protocol (an attempt to fully understand every step in process to make it easier when I actually conduct the extraction), I noted this:

• SDS/Sarkosyl lysis buffer
3 volumes of 4% SDS buffer
1 volume of 5% Sarkosyl Buffer
5 μg/mL proteinase K
0.12% of β-mercaptoethanol
Add 200 μL of Proteinase K (2Omg/mL) and 100 μL of β-mercaptoethanol to 60 mL of 4% SDS buffer, and 20 mL of 5% sarkosyl buffer.
Do not refrigerate or autoclave. 

It appears as though there is some conflicting information (high-lited in blue). The first 4 lines of the quote above are like the ingredients of the buffer, and the sentence beginning with add is like the recipe of how to make it. Five micrograms per milliliter and twenty milligrams per milliliter are vastly different concentrations! I could understand if the first concentration given was higher than the other - that would indicate that I would need to make a dilution of it in order to conform to the "recipe." How exactly would I get from a lower concentration to a higher though? I consulted with Josh and Matt and they were also unable to make sense of this either. Our consensus was for me to find other protocols that use proteinase k and compare concentrations.

Just when I thought I was done digging through protocols... que sera sera...

To date, I haven't delved into this fully. It does appear that 20mg/mL is a common concentration for proteinase k. I will look into this more as I want to be certain so the potential for error is reduced.

New Protocol Redux

Last week I spoke about being very happy with a new extraction protocol that I had found. Scratch that. Upon further investigation into it, I relised that the protocol listed all of the chemicals required for each of the buffers used, but failed to itemize the ratios of these chemicals in the solutions. Essentialy, I had the ingredients without the recipe.

After several more hours of digging I came upon a great resource website: http://www.protocol-online.org. Through this site, I came upon an outstanding list of DNA extraction protocols from the University of Wisconsin-Madison: http://labs.medmicro.wisc.edu/mcfall-ngai/papers/2002nish3.pdf. From this list I will be using protocol 17, sans the liquid nitrogen. The "outstanding" part of this list is the detailed list of exactly how to make many of the common reagents for DNA extraction. The shopping list begins.

Last Friday I did make it back out to my sampling site at Squaw Peak and collected fresh specimen from the same subjects I have been working with. I was more than a bit worried that the recent monsoon activity would negatively impact my field location as it is a wash. At first blush, there did not seem to be any major changes, but I was unable to observe the site closely as I was pressed for time. I will be returning in the not to distant future to more closely examine the site for changes (are more of the root systems exposed showing the relation between individuals). I spent a little over an hour collecting leaf samples, washing them in ethanol, and labeling them. They are currently in storage at -80 degrees Celsius.

A new protocol

I have found a new protocol for DNA extraction that uses a couple of elements that I have not used before. This protocol from Plant Molecular Biology Reporter seems straightforward enough, and uses a chemical, hexadecyltrimethylammonium bromide CTAB, will alleviate interference from various metabolites present in plants. It also uses a chloroform:isoamyl alcohol which separates proteins and polysaccharides from the nucleic acids in order to yield a cleaner extraction. After looking rather extensively at my results from last semester, I feel that my main issue was not being able to obtain a clean genetic extraction. Keeping this in mind, I will also be obtaining new leaf samples (from the same subjects as before) and adding a step when collecting them as well. When each sample is obtained, I will give them a quick wash with ethanol to remove any pontential environmental contaminants. 

Tomorrow I will go through my laundry list of required reagents and see what the lab has on hand, and what will need to be ordered. If time permits tomorrow, I will head out and collect the new samples. If I cannot do this tomorrow I will most likely do so over the weekend as I want to get started with extractions as soon as possible. I can already feel the clock ticking on this semester.

Another beginning or a continuation

Ah fall semester here you are again.

This semester I will be continuing my research with our friend the good old Palo Verde. Through my previous research I have very good observational evidence that the Palo Verde does reproduce through cloning in addition to it's typical sexual reproduction. I also attempted to show this through the comparison of the genetic makeup of individuals suspected of being clones. Unfortunately, last semester I was not able to produce a viable genetic extraction protocol for the Palo. It is my hope that this semester I will be able to perfect my extraction methodology and produce good samples for PCR amplification (and later comparison). I already have several ideas for cleaning up my extractions and will begin looking for better techniques to use.

I also would like to delve a little further into the ecology of the desert wash as well. In my observations of the Palo in the field, I noticed several very unique characteristics of the wash environment and would like to explore them in a little more depth. Specifically speaking the composition of the soil layers.

So, here's to another adventure in science!

The end... again...

Well, here we are at the end of the semester again. As I write this, my comrades-in-arms of the S-STEM program are at the Estrella Mountain Community College campus getting prepared for their poster presentations at the student conference. Unfortunately, my research was not accepted for presentation at this conference. In all actuality, I am happy about this. Due to failures in being able to produce good results with my genetic extractions, I would have only been able to present evidence based on the observable characteristics of the Palo Verde rooting systems. Even though this is very good information, it would have felt like I was only presenting half of my work. That does not sound like a big deal, but to those that know me, it kind of is. I feel that there is a lot of potential in this project and I want to see it to its end and be able to present the totality of the work. I hope that I am lucky enough to still be involved in this program next semester to be able to conclude (successfully, I hope) this research.

Having said all of that I wish the best for my fellow stemmers presentations today and look forward to seeing everybody's work displayed/presented tomorrow in the lab. Speaking of which, I need to put the final touches on my PowerPoint presentation for tomorrow, so I will leave you with this:

Credit unknown, my wife sent me this in jest. 

I thought it poignant. It seems after all the design and preparation involved in a presentation, nobody ever really considers how to end it. This can lead to a very awkward and uncomfortable moment... :)

Good luck everybody

Hope to see you all next semester!

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.