Plan B

In the previous blog I talked about the main goal of my project; to find the most beneficial way to extract DNA from bacteria that will run in two different techniques; gel extraction and in the  PCR (polymerase chain reaction). In the last experiment I was successful in running the gel extraction with three bacterias; Salmonella, E. Coli, and S. Aurues, but not successful in the PCR technique.
The next day after finding out that the PCR did not work, it was time to test a different protocol. This protocol is called "Isolation and Purification of Total Genomic DNA from E Coli", an experiment that is longer compared to my first experiment.
The procedures for this experiment began just like the first experiment. I had to grow bacteria over night for 24 hours in a TSB tube, but since I had my bacterias already growing from the first experiment I went ahead and used the same ones (E. Coli, S. Aureus, S. Epidermidis, and Salmonella). My S. Epidermidis tube was spilled when transferring so I used a different TSB tube that was grown a week earlier. I had to do some centrifuging at high speeds for intervals of 2-3 minutes. As well as some incubating at 37-80 degrees Celcius for 30 minutes and 5 minutes. Lysis solution was added to disrupt membranes and denature proteins. RNase solution was added to degrade RNA into small fragments or ribonucleotides. Protein Precipitation Solution to add insoluble material for the centrifuging step. Isopropanol was added to the solution, also 70% ethanol, and lastly the DNA Rehydration Solution (TRIS EDTA Buffer). In the end of all these procedures the extractions made were ready to run in either  techniques. The first one I did was the gel extraction, the gel was made out of 100 milliliters of TAE and 1 gram of molecular Biology Agarose. When analyzing the gel under uv light, I came to the conclusion that this extraction only worked for the E. Coli and not for the rest. Once again a PCR was not done because I had no DNA from the extraction to run. Here are some pictures enjoy !


Closest to 80 degree Celsius
the incubator reached.  

Gel Extraction Apparatus

TAE Solution

 

Lets do this !

After a week of deciding what my project would consist of, DNA Extraction came to the rescue. I was introduced to the Extraction of Bacterial DNA from Gram-Positive and Gram-Negative species experiment. The goal of this project is to find the best method to extract DNA from various bacteria that will efficiently and successfully run in the PCR (polymerase chain reaction). The PCR is a machine that is used to amplify either a single or a few copies of DNA resulting in the duplication of  several, to millions of copies of a specific DNA section. However to actually run a PCR we have to first extract the DNA and prove that there is DNA present to run it, most of my time was dedicated to this first step of the project. In order to find out if DNA is really gained from the bacteria extraction a gel extraction technique was used to prove it. The gel extraction is a different type of technique that isolates the DNA from an agarose gel, and with the help of uv light we know if DNA is present from the extraction done.

The first protocol I tested was short and simple and glad to say successful. Just a few steps were required as opposed to the second protocol which was more complex. For both, I made a culture for four different bacterias: E. Coli, S. Aureus, S. Epidermidis, and Salmonella. I placed a drop of each into their own TSB (tryptic soy broth) tubes and allowed the bacteria to grow for 24 hours. The next day I noticed that the S. Epidermidis did not have any bacteria growth, compared to the other three. I had to centrifuge each of them for 3 minutes under 5000 rmp, then extracted the TBS. I washed the bacterial cell pellet with.5ml sterile water following up with 3 minutes in the centrifuge. After the pellet was washed I extracted the supernatant and  added 100microliters of QuickExtract Bacterial DNA Extraction Solution. With that I added 1 microliters of Ready-Lyse Lysozyme Solution to each of the tubes. After a few minutes of incubation at room temperature the tubes were ready for the gel extraction and the PCR.


        

Test tubes
Top Row Ready for Gel Extraction
Bottom Row Samples from TSB culture

 

 

Left = QuickExtract Bacterial DNA Extraction Solution
Right = Ready-Lyse Lysozyme Solution


 

My results for the gel extraction were great ! Except for the S. Epidermidis, because just like I suspected the DNA extraction was not successful. You can see this by the really dark square next to the E.Coli. That same day left the PCR running with the salmonella but unfortunately the next day I found out that the PCR test did not work. This conclusion has left me with the second protocol to test. I will soon have results of which protocol gave the best results, and which one is able to continue with the PCR.                                                                  



Gel Extraction under UV Light

First Row                   Ruler

Second Bright Row    E.Coli

Third Dark Row         S. Epidermidis

Fourth Medium Row  S. Aureus

Last Bright Row         Salmonella

Strokes: Therapy, Drugs, & more

 
Restorative Neurology “Drugs and Recovery Following Stroke”

This past week I have dedicated most of my time to an article I found that's main focus was the recovery process of a stroke. The main topic for this journal dealt with four main points, the biological and environmental factors of a stroke, the responses to the injury, neuronal rearrangements, and the adaptive responses.

Thanks to laboratory work, these four factors can now be understood and also provide information on the recovery process for a brain injury such as the stroke.  The way these researches went about their experiment involved four different procedures. Investigations were done for the biological and environmental factors, this was more of an observational study which involves a simple task such as book keeping, and keeping an open eye for changes that stood out. Some of these details that  were compared to each other were for example the size of the specific injury, the location, the rate this injury occurred at and how fast these injuries took to recover. For the responses to injury section and the adaptive responses experiments were done on animals to compare the variety of responses the brain had to different scenarios. Cerebral edema is an example of the most common response, which is the excess accumulation of water in the intra/extra spaces in the brain. These responses were also compared in different areas, how fast they took to respond, how beneficial these responses were, and how they react to a variety of drugs. Taking to note that out of all the drugs that were used the two that are normally prescribed now on humans, actually were the ones that slowed the recovery process even more, these two were phenytoin and benzodiazepines. The third section also involved rats mostly adult rats. The neuronal rearrangements dealt with the new connections our neurons and axons make after an injury occurs and the connections that are lost. Some of these connections appear to happen 8 hours after an injury which would be beneficial if these connections would help, but they seem to impede the recovery process. In the other hand the connections that would be beneficial are of course the ones that take months to happen.

There are many drugs out there at this very moment that seem to aid the injury, however none stand out from the rest. As a person interest in neurology and someone who had her grandpa suffer such an injury I actually found this article very interesting, and even if you don't think this would interest you try and learn a little about it.  I believe we all can agree the brain is the reason we function making it very important, but yet we still do not understand how it truly works.

Here is the link if you are interested in reading more.
http://stroke.ahajournals.org/content/21/11/1636.short

Citation
Goldstein, L. B., & Davis, J. N. (1990). Current Concepts of Cerebrovascular
     Disease and Stroke. Restoratiave Neurology: Drugs and Recovery Following
     Stroke, 21, 1636-1640. doi:10.1161/01.STR.21.11.1636