Week 9, Semester 2.

Colorized low-temperature electron micrograph, E. coli. https://www.flickr.com/photos/microbeworld/5981923914/

Welcome back to this blog!

This week's focus consists of running a polymerase-chain reaction (PCR) on the DNA samples previously discussed in the Week 5, Semester 2 edition of this blog. It remains to be seen if the primers utilized were successful at the DNA amplification process, for as of publication time the PCR was still in progress; thus the results had not been analyzed.

The next phase of this project will proceed beginning next week with the extension of the extraction protocol to other bacterial species endemic to this lab. The methodology utilized for E. coli extractions and testing will be repeated with each additional species; every phase of the project from extraction, to electrophoresis gel, to spectrometer reading, to finally PCR will be repeated with each additional species to identify any correlations or errors in the methodology used and results obtained. 

After the multi-species extractions have been finished, the project will conclude for the semester with an analysis of the genomic sequences specific to each bacterial species. The primers selected for the initial E. coli PCR's were referenced as being universal in the work Critical Evaluation of Two Primers Commonly Used for Amplification of Bacterial 16S rRNA Genes (Frank, et. al, 2008) and were therefore selected for further testing. However, it is hypothesized that further examination of the individual genomes is warranted to determine if a new primer design is needed for application to species other than E. coli.

Further details to follow on the next blog issue.

Week 8, Semester 2. Success!

Hello! And welcome back to my blog.

This week, I resumed testing of samples previously extracted from E. coli bacterium. Both protocols submitted for analysis utilized a 0.3 ml TBS and 10 µl SDS solution to lyse the cells, a 55°C heating and ice cooling method combined with a protein precipitation solution to reduce protein contamination and an isopropanol/ethanol wash to precipitate chromosomal DNA and clean the DNA pellet.The protocols differed in the substances used for protein precipitation; protocol one (P1) utilized proteinase K, and protocol two (P2) utilized a guanadine hydrochloride/sodium acetate solution. They also differed in length of precipitation in isopropanol; P1 was in solution for five minutes while P2 remained in solution @ -20°C for five days.

The samples were placed in an electrophoresis gel for analysis. The gels were created @ a ratio of 20 g agarose/100 ml 1 X TAE buffer ratio, with 4 µl of 10,000x concentration SYBY green DNA stain added to solution post-heating.

The spectrometer was also used to measure ultraviolet absorbance ratios of the four samples. Samples were subjected to 260 nm, 280 nm, and 320 nm consecutively.

Results:

DNA!

All four samples showed banding in the gel; P1 sample two contained the most clearly visible banding. The photo that follows is of the samples. The clarity is a bit off due to the age of the camera used to capture the image.

Calculations from the spectrometer readings to follow on my next blog. Happy learning!

Week 7-Semester 2.Mathopolis

Hello! Lab work is progressing, albeit at a slower pace than I would prefer. However, previous efforts to accelerate the pace of my project have shown that a consistent, repeated, and measured approach is most conducive to accurate results. Whether double- and triple-checking all calculations before beginning a protocol, or repeatedly verifying the settings on a pipette as I work, I have found that adhering to an established technique is the key to both finding and repeating accurate results.

Luckily for me, I have been inundated by the structured world of math this semester. I am taking Chemistry, Physics, and Trigonometry concurrently, so I spend quite a bit of time weekly honing my mathematical skills. This focus has transferred over to my research here in the lab and increased my proficiency at performing the calculations needed to move forward with my project.

This week has been heavily focused on gaining and retaining math skills, but I did focus on extractions as well. I was also able to finally utilize the recipe for the protein precipitation solution provided by Cori Leonetti, our in-house microbiologist. Unfortunately, due to a scheduling conflict with some classes, I was unable to access all the equipment needed to complete my protocols until late in the week. Therefore, the results of this week's work remain to be seen; until the process has been completed, here is the solution I am using to precipitate proteins from my DNA samples.

4.2 M Guanidinium Hydrochloride
0.9 M Sodium Acetate
4.8 pH target
De-ionized water to reach total volume of 50 mL

I calculated the molarity of the solution targeting a final volume of 50 mL. The GuHCl was in an 8.0 M solution; the sodium acetate was in solid form, so I used a mole-mass conversion to calculate the amount needed. I then mixed, observed for solubility, and added de-ionized water until nearly at final volume. I then measured the pH. The result was approximately 6.3, so I added HCL by dropper until the pH was reduced until it reached the desired result. I then added the remaining de-ionized water to achieve full volume and re-measured the pH for a final result.

More on the usage of this solution and further extractions to follow. Until then, please enjoy the following:

Week 6-Semester 2. Reference Guide #1

Hello! This week again finds that my DNA extractions and subsequent absorbance readings conducted using the spectrophotometer will fall on Friday, the day after this blog is due. As a result, any data collection and methodology conducted this week will appear in a later blog post.

Part of my research project this semester is to become personally proficient in the use of all lab equipment and also guide any students that follow in the operation of the previously mentioned (on this blog) “lost” spectrophotometer by compiling detailed instructions for usage. Therefore, this week’s post will be a brief process analysis about using the Helios spectrophotometer to take live UV absorbance ratio readings.

The reader should be cautioned that there are additional methods for taking readings, such as programming and saving fixed methods, and other nuances within the settings menu that may affect sequence progression of individual wells and absorbance ratio levels. This additional information will be included in future blog posts. In the interim, I have pre-set and saved the settings needed to conduct the following protocol. If implemented as described, without any changes to the settings menu, the user can quickly and easily take accurate live absorbance readings.

Quick guide to live reads:

Notes/overview:

·         The Helios needs 10-15 minutes to warm up before being used.

·         The user should not leave the lid open or peer into the bay for an extended length of time; safety goggles with UV protection are recommended.

·         Bacterial DNA solutions should be at a 1µl DNA/99µl diluting liquid ratio (total volume 100µl). Molecular biology-grade water works best due to its purity.

·         The control sample should be 100 µl of the same liquid used to dilute the DNA sample.

·         Due to an inherent flaw in the design of the Helios, both control and DNA samples may need to be increased to 200µl total volume each to improve data collection.

·         One reading will be taken of the control sample @ 260 nm to set a baseline.

·         Each DNA sample will be subjected to three nm readings in the following order: 260 (to measure DNA presence), 280 (to measure protein contamination), and 320 (to measure turbidity, i.e. additional contaminants). It is important to begin with the 260 nm setting, as any DNA will be degraded by the higher wavelengths, and accurate measurements will be negatively affected by using a different progression sequence.

·         The user should record absorbance ratios given by each nm level in order to later calculate DNA purity and concentration.

Begin:

1)      Insert control cuvette into well #1. Place cuvettes containing DNA samples in wells 2-7 as needed.

2)      Make sure well #1 is aligned in anterior position (between UV bulbs).

3)      Program control sample:

a)      Set UV nm @ 260 nm for zero-base reading by pressing “nm” on LED display.

b)      Type desired nm wavelength (260) in display. Hit enter.

c)      Press zero-base key to set read at zero.

d)     LED display should read 0.000 A 260 nm. Record data by writing it down (or print by pressing print key on LED display.)

4)      Program DNA samples:

a)      Use right arrow key to advance to desired well.

b)      Set initial read @ 260 nm as outlined in step 3.

c)      Display will show absorbance reading @ 260 nm. Record data.

d)     Re-set nm @ 280 nm. Record data.

e)      Re-set nm @ 320. Record data.

f)       Repeat steps 4a-e for additional wells.

The procedure is as simple as that! Good luck with your samples!

Please enjoy this excellent website from the Smithsonian about DNA. Website is also photo credit:

 http://www.genome.gov/smithsonian/ 

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