Week 14-Semester 3. Data duplicated.

Hello! Welcome back to my blog.

This week has been devoted to getting ready for the end of the semester, but I did manage to run a couple of gels on my PCR samples from last week in order to verify the results I obtained.

I am glad to report that this latest round of gels showed that DNA amplification did occur in 8 of the 9 bacterial species I tested. This is a slight change from the 9 reported last week, but still an excellent result. The reason for the discrepancy in that one organism? I suspect the original well correlating to that species that showed DNA may have simply had some bleed-over from an adjacent well. This shouldn't happen in a perfect scenario, but in a real-life lab situation, odd things and errors occasionally happen.

At any rate, this result does not yet indicate that amplification is not possible for the species in question, Pseudomonas aeruginosa. Pseudomonas differs slightly from the other organisms I am testing, in that it forms biofilms which can impede DNA extraction from the cell. In fact, during the extraction process that produced the series of samples I tested for this experiment, I did not see visible chromosomal threading during the alcohol precipitation phase of the extraction. While a lack of visible threading does not automatically indicate the absence of DNA, it is a possibility that cannot be ignored. Therefore, I must adjust the extraction protocol for the presence of biofilm, conduct another extraction, and re-run the PCR experiment on Pseudomonas a couple of more times in order to rule out the possibility that my first round of experiments may not have produced any DNA for amplification.

Such additional tests, however, will have to wait until the summer session resumes, as our last week of lab time is upon us.

Until then, have an excellent end of the semester. See you soon!

Image credit: imgbuddy.com

Week 13-Semester3. A Breakthrough!

Hello! Welcome back to my blog! Good news to report in this edition.

Last week, I had some initial success amplifying DNA from S. enterica, S. sonnei, and K. oxytoca using the first set of primers I had previously designed. As you may recall, I conducted a PCR reaction that showed positive results (for DNA amplification) and then repeated the experiment using a different set of DNA samples (in order to repeat the results and establish a pattern of amplification). Based on the data from those PCRs, this week I expanded my experiment to include the other organisms in my study. Using the same PCR protocol and primer set, I set out to see if amplification of the other species was possible.

The result? Of the nine species identified for this project, a total of five amplified during the third PCR. I considered this to be promising, as the fact that there was amplification indicated a number of things:

• The technique used to identify nucleotide sequences in the gene for potential amplification worked.
•  The annealing temperature parameters selected for the tested primers worked. 
• Given that amplification was demonstrated to be possible, testing of the other primer sets designed for this study could proceed.

This in itself was promising news. However, I still needed more data for my research paper, so I decided to expand my testing to include the next set of primers in the queue.

Therefore, I immediately set about running another PCR. I duplicated all controls from the first three reactions, exactly; even the annealing temperature remained the same, due to a similarity in optimal temperature ranges between the two sets of primers.

The results of this new test? Success! All nine of my organisms showed DNA banding in the post-PCR gels. Meaning that for the first time and after a year of testing, I was able to amplify DNA from all of my target organisms using one primer set of my own design. This, of course, is data that made me quite happy.

Also of course, this data is only preliminary; I will need to re-create the results a couple of more times, using a molecular marker, before I can be certain of the results' validity.

Looks like I know what I will be doing for the rest of the semester!

Until next week, cheers.

Paul C.

PS: Here's a link to a video about potential cloning using Mammuthus primigenius DNA.  http://www.sciencedaily.com/videos/5afc2e6cc20523ee3fc44352717bb039.htm

Photo credit: a-z-animals.com. The wooly mammoth in the collection of The Royal BC Museum, Vancouver, BC.

PPS: For more details about the work I have done this semester, including protocols, supporting data, and gel images, my research paper will be available for review via a link on this blog at the end of this semester.

Week 12-Semester 3. Finally, PCR results.

Hello! Welcome back to my blog.

This week, I was able to get lab time on the thermocycler, and thus began the final phase of my project. This last portion involves testing the nucleotide primers that I have designed for universality against the nine bacterial species selected for this project. I have already successfully tested a number of Universal Rice Primers against my extraction samples, so all that remains is to test these new primers in various combinations and at a number of different annealing temperatures and then analyze the data that provides.

Theoretically, the series of primers I am testing should successfully amplify DNA in three of my samples because the nucleotide sequences they contain were developed using the genetic sequences of those three bacteria. Therefore, I ran the first PCR of this project phase on the species S. enterica, S. sonnei, and K. oxytoca. I used an annealing temperature of 64°C during the reaction, which was squarely between my optimal primer temperatures of 62.4°C and 68.7°C. The results? DNA! However, the clarity of banding was extremely poor for S. sonnei and K. oxytoca. This may have been an indication of a number of things, including the amount of DNA present in my original extraction sample, the annealing temperature used, or the primers themselves.

In order to rule out/identify my sample itself as the potential culprit, I ran another PCR reaction using DNA samples, from a different extraction date, that were previously amplified using Universal Rice Primers. All controls in the reaction were an exact duplicate of the first PCR I ran this week.

The results? DNA, clearly visible for all three bacterial species, in my post-PCR gel.

This is a success for me on two fronts. One, it proved my hypothesis that my sample was responsible for the results of experiment #1. Two, it showed that the primers I designed work. This makes me quite happy, for it is an affirmation that my rudimentary understanding of genetics is improving.

Next week, due to these promising results, I will expand my PCR reactions to include the remaining organisms in this study. Until then, enjoy yourselves..and enjoy the following information about Neanderthal DNA that was recently extracted from an Italian specimen.

http://www.livescience.com/50458-oldest-neanderthal-dna-found.html

The above links to background information about Altamura man, a specimen of Homo neanderthalensis found in a cave in Italy approximately 150,000 years after his death.

This image shows the stalactites covering the specimen. Source: earth66.com 

Week 11-Semester 3

Hello! And welcome back.

There has been an ongoing issue these last couple of weeks with getting access to the thermocycler, so any plans to proceed with my project are on hold until that runs its course. My project has progressed to the point where all I need to do is collect data from PCRs so that I can evaluate the various primers I am using to amplify DNA; without the ability to run PCRs, all I can do is repeat what I have already done with extractions and basic lab methodology. Therefore, I have no data or conclusions to report this week.

This is actually a propitious development, as we are now nearing the last four weeks of the semester, the time when we all get slammed by course work. So although I am saddened that I cannot run reactions this week, I have instead been able to re-direct that time towards catching up assignments. This extra time is also providing a bit of relief for me because this has been my busiest semester to-date. Between the various jobs I have and the demands of academia, I have had about three days off total this entire semester. But lest one think that I am complaining, let it be known that I am grateful to be able to go to school and grateful to be in the S-STEM program.

That's it until I have some data to report. Have an Excellent Week!

P.S. Here is a picture for you, just because puppies always make people happy:

Photo credit: www.hdwallpapersinn.com

 

Week 10-Semester 3

Hello! And welcome back to my blog.

This week, I spent a great deal of time refining my extraction protocol to account for the increased growth encountered after I switched mediums from luria broth to TSB. I adjusted the initial sample size used to create a pellet for the extraction from 4.5 mL to 0.5 mL, and and reduced the amount of time that I centrifuged and vortexed each sample during the protocol application by 50% or more. This resulted in viable DNA samples for five of my eight bacterial species; the remaining three did not produce DNA. Because of the late date of the semester and the fact that I need additional data before I can write my research paper, I have decided to switch back to the growth medium I was previously using successfully for all eight species. This will allow me to get the data I need without losing any more time tinkering around with a basic procedure.

I also was able to spend some time running a PCR on one of the primer sets that I previously designed and have been excitedly waiting to test. Unfortunately, and to my profound disappointment, I have decided that the data generated from that experiment is unusable due to an error in the protocol used to provide the DNA sample used in the reaction.

I am going to re-test the reaction using another set of DNA samples that have already been evaluated for DNA using an electrophoresis gel. I have been storing the samples on ice for just such an occasion as this. The samples have been repeatedly subjected to gels, and have showed clear banding every time for all eight target species.

Sometime next week, I will set up a reaction using those samples and the primers I have designed. In the interim, please have an excellent weekend.

Please enjoy this photo of Proteus mirabilis, one of the species I am using. This little guy is commonly found in the human gastrointestinal tract, soil, and water. It can cause a number of infections in humans, and has been linked to the formation of kidney stones. (Davis & Zuber)

Photo credit:  CDC Public Health Image Library. CDC (PHIL #6691), 1976.
Link for more information from Davis & Zuber:  http://www.clinicianreviews.com/specialty-focus/nephrology/article/iproteus-mirabilisi-isolating-a-cause-of-kidney-stones.html