Week 5, Semester 2. 10/01 Success!

Hello!

Last week, after learning the nuances of the lab spectrophotometer, I was able to run my previous week's extractions through the spec and get some readings. According to the data collected, I was successful at extracting DNA. This is excellent news, as far the progression of my research is concerned.

I was quite surprised by the differences in the amount of DNA successfully collected and what materials provided those results. The methodology utilized in both of my extractions was identical, only varying in the type and amount of protein precipitation solution I used to clean up the sample. I used an expensive proteinase K dilution ($40/ 5 mg vial, diluted @ 5 mg/250 µL) for protocol number one and an inexpensive (99 cents, diluted at 0.05 g/µL), commercially available meat tenderizer dilution for protocol number two. I had expected the proteinase K solution to realize the best results, yet instead it was the cheaper solution that produced the most DNA. Protocol number one only showed a DNA concentration of 15 µg/mL, while the cheaper protocol number two yielded 1535 µg/mL. This was a substantial difference that only reminded me that, to date in my research, the simpler methods are often the most useful.

I am still searching for a DNA sample of known concentration that we have in-stock, so that I can dilute it and take a spec reading on it. Since the concentration will already be known, I can use it as a control to verify that the measurements I am taking on the spec are in fact correct. More on that to follow as this project progresses.

Until next week, cheers! And enjoy this Ted talk from one of the Nobel Laureates who discovered the structure of DNA, Dr. James Watson.

http://video.ted.com/talk/podcast/2005/None/JamesWatson_2005-480p.mp4

Week 4, Semester 2. The Spectrophotometer

Greetings! This week's lab time has been devoted to perusing instruction manuals and searching online for information about the Helios spectrophotometer located in the lab. This is an excellent device that can be used to measure the ultraviolet light absorbance ratios of DNA extraction samples. The machine is a high tech and more accurate version of electrophoresis gels; both methods can tell the operator if there is actually DNA present in the sample measured.

In a nutshell, the spectrophotometer operates as follows: One loads the sample, and then takes measurements using different wavelengths of ultraviolet light. A wavelength of 260 nm will indicate the presence of DNA, a wavelength of 280 nm will indicate protein contamination, and a wavelength of 320 nm is used to measure the turbidity of the sample, i.e. other possible contaminants. From these measurements, the operator can then use mathematical formulas to calculate the DNA purity and DNA concentration of the sample. This allows the operator to determine if his extraction protocols have been successful at removing DNA from his source material.

The Helios spec that we have in the lab was originally purchased more than a decade ago, and subsequently the knowledge needed to operate it has been lost. Therefore, I've spent most of my time this week educating myself as to the nuances of this particular brand. It's been a bit of a search, as the model was discontinued in 2011, and online technical support is no longer available. We still have the instruction manual, but it has little more information in it other than programming. Nothing is available in the manual about actually installing reading protocols. However, with a little diligence I have figured it out and am prepared to now use it to measure my samples.

Therefore, the remainder of my lab time this week will be spent extracting DNA from my E. coli cultures and then running them through the spectrophotometer to verify the protocol's success. More on that methodology and any successes or failures will follow on this blog next week. Until then, please enjoy the photo below of the machine that has taken all of my attention this week.

Cheers!

bioorganickachemiesite.upol.cz

Week 3, Semester 2. A New Path

Last week I prepped extraction samples from E. coli, using a simple extraction protocol provided by the lab's resident MicroBiology guru, Cori. Apparently, sometimes the simplest methodology is the best, as once the extractions were complete, I ran electrophoresis gels on the samples. The results under UV review indicated that DNA was successfully extracted. Hooray! (Or should I say, "Eureka! I've got it!") Exact details of methodology are available upon request, but as a general overview the protocol simply used TBS and SDS to lyse the sample cells, and room temperature isopropanol to precipitate the DNA. I then stored the samples @ -4°C for further testing this week.

This week's major breakthrough in my learning process occurred when I decided to figure out how to operate an old, unused photo-spectrometer that has been sitting in the lab for the better part of a decade. While discontinued from production, the unit isn't nearly as antiquated as one might think, and is decades newer than some of the other photo-specs I found stashed around the lab. It has a digital display for entering scanning protocols, and comes equipped with an internal printer to provide a hard copy of the test results. 

In addition to learning the operations of this piece of equipment, I also refreshed my knowledge of UV wavelengths and the optimal range for UV scanning of DNA without sample degradation.
I did run some preliminary samples through the spec, but due to a calibration oversight the data was incorrect and will have to be repeated. Results on this to follow next week.


Here's an artist rendering of DNA. Enjoy!

Source: deviantart.com

Week 2, Semester 2

Today I prepped for bacterial extractions, which begin in earnest tomorrow. To that end, I spent most of my time in the lab reviewing previous data results for potential improvements and/or changes to procedures, as well as taking inventory of the supplies I will need to complete the extractions. I also did mundane but necessary tasks, such as creating fresh buffers and gels for future electrophoreses.

I have identified two protocols that I have previously tested for further review. However, I will be changing the protein precipitates that I use to clean up the sample. A proteinase K solution and commercially available contact lens cleaner that I used in previous protocols provided promising results, so I will be testing them with the different protocols to determine if I can repeat or improve upon my previous data.

Due to the time needed to precipitate the DNA samples. I will be leaving them to incubate @ -4° C over the weekend. Hence, I will be unable to conduct a PCR to identify any successfully extracted, non-sheared DNA, until I am in the lab the following week.

Results will be posted on this blog at that time. In the interim, please enjoy this rendering of E. coli in glass, by the artist Luke Jerram.

http://www.lukejerram.com/glass/

Week 1, Semester 2. Summer Recap

Hello! The last post on my blog left off as the S-STEM Scholars were getting ready to present their research results at the Estrella Mountain Student Conference. I am happy to update any incoming S-STEM Scholars with the following news: The Phoenix College Bio-Sciences Department swept the awards! Our students took all prizes in the visual presentation category. Kudos and congratulations to the prize winners. The accolades are well-deserved (and so were the prize monies that were distributed to the winners!)

Summer 2014 was utilized to continue variations of my spring research project. I continued to conduct DNA extractions on E. coli specimens, using multiple protocols and a variety of protein precipitates within each. Results were varied, with limited success. All samples were processed through electrophoresis gels and then viewed under UV light to determine banding, if any. Only two samples showed promise at this step, but when I progressed to polymerase-chain reactions (PCR) on those samples, the results changed from inconclusive to negative.

The semester was not fruitless, however. As I continued the processes of extractions, electrophoreses, and PCR's, my knowledge and expertise improved significantly. My fundamental understanding of DNA composition, lab equipment, mathematical calculations, the scientific method, and even my note-taking proficiency, were all improved by the extra fifty hours I was able to spend in the lab. Indubitably, this increase in knowledge will lead to positive results as I continue my project over the fall semester.

After a brief hiatus from my project that I have taken as summer became September, I am looking forward to resuming my work, re-connecting with old friends, and meeting the new scholars as we embark on another learning journey. See you in the lab!

P.S. For any math scholars out there: I would love to discuss summing integers and digital roots with you. I have been studying the phenomena of multiples of 9 whose integers sum back to 9; After multiple calculations and conferencing with Josh James, I became aware of the reduction patterns that accompany whole numbers from 1-8 after summing as well. This mathematical concept is absolutely intriguing me at this moment, and any light you can shed on the subject would be welcomed. Feel free to contact me!

credit: sapnaedu.in