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