Last week I worked on my extractions. I did about three extractions using the same protocol and the same bacteria. If you have been following up with my experiments you now know that the last extraction was definitely the best I have done and the results from the gel electrophoresis can prove it. Now that I have mastered the extraction part of my experiment running a PCR for each bacteria should be easy.
Let me remind you that PCR stands for Polymerase Chain Reaction, and what this type of machine or technology tool does is it takes a piece of DNA and amplifies it by making several thousands to millions copies of a specific DNA sequence. This makes large and efficient amount of DNA that could be used for several tests. For example finding out suspects from a criminal act, finding out who a deceased body belongs to, or track down virus and diseases. However in my case I am just running this experiment to see how successful I can be.
In order to even begin a PCR I must have the DNA of the bacteria extracted and ready to go, because I used E. Coli, Bacillus, S. Aureus, and Agrobacterium, I continued with the PCR protocol from these same extractions. The PCR works a little different from the gel electrophoresis because for one it takes six times longer! We add different reagents and components such as master mix, primers, DI water and of course DNA, the order in which we mix these four components and reagents together is important and when doing this we must keep them on ice. Primers are reagents that contain nucleic acid and are included in this mixture because this nucleic acid helps start the DNA synthesis. The master mix is a reagent that contains Taq polymerase, and this polymerase helps amplify short DNA segments. The addition of water helps dilute the concentrations of both reagents just mentioned and DNA has to be included. I ran two PCR the first one was with primers 4 and 5 with agrobacterium and E. Coli. The second PCR I tried was with primers 3, and 4 with E. Coli, S. Aureus, Bacillus, and Agrobacterium. To check if the PCR produced DNA I run a gel electrophoresis just like I run it for the extraction part. I was not successful with the first PCR and failed to capture a picture of it, but the second try was a lot better.
Let me remind you that PCR stands for Polymerase Chain Reaction, and what this type of machine or technology tool does is it takes a piece of DNA and amplifies it by making several thousands to millions copies of a specific DNA sequence. This makes large and efficient amount of DNA that could be used for several tests. For example finding out suspects from a criminal act, finding out who a deceased body belongs to, or track down virus and diseases. However in my case I am just running this experiment to see how successful I can be.
In order to even begin a PCR I must have the DNA of the bacteria extracted and ready to go, because I used E. Coli, Bacillus, S. Aureus, and Agrobacterium, I continued with the PCR protocol from these same extractions. The PCR works a little different from the gel electrophoresis because for one it takes six times longer! We add different reagents and components such as master mix, primers, DI water and of course DNA, the order in which we mix these four components and reagents together is important and when doing this we must keep them on ice. Primers are reagents that contain nucleic acid and are included in this mixture because this nucleic acid helps start the DNA synthesis. The master mix is a reagent that contains Taq polymerase, and this polymerase helps amplify short DNA segments. The addition of water helps dilute the concentrations of both reagents just mentioned and DNA has to be included. I ran two PCR the first one was with primers 4 and 5 with agrobacterium and E. Coli. The second PCR I tried was with primers 3, and 4 with E. Coli, S. Aureus, Bacillus, and Agrobacterium. To check if the PCR produced DNA I run a gel electrophoresis just like I run it for the extraction part. I was not successful with the first PCR and failed to capture a picture of it, but the second try was a lot better.
PCR Steps
1. Add 10 microliters of master mix
2. Add 5 microliters of DI H2O
3. Add 4 microliters of primer
4. Add 1 microliters of DNA Sample
1. Add 10 microliters of master mix
2. Add 5 microliters of DI H2O
3. Add 4 microliters of primer
4. Add 1 microliters of DNA Sample
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| Primer4 E. Coli, S. Aureus, Bacillus, Agrobacterium |
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| Primer 3 E. Coli, S. Aureus, Bacillus, Agrobacterium |
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