The End

This week was a stressful but successful week. I say this because I was able to finish my project for the ASU conference which is only a week away! But I have to say it was tricky trying to get all my information neatly organized. Plugging the information was the easy part of this project, but making it all fit took so long. Playing with the colors, font, font size, and all the effects kept me entertained for a while. I was able to finish this over the weekend and on Monday when I came in Matt nicely went over it and gave me some feedback. I was able to change a few things to make it better and even though I personally like the pink color I originally chose that  looked cute, I changed it to a lighter pink because the one I had was too bright. Anyway, since my project is finished and Matt has already gone over it I want to see if all of you would be willing to give me some feedback on my project. It is in our dropbox for all of you who want to check it out.

Since my project is done with I think it is only fair to share with you what my conclusions were. Let me catch you all up with what my purpose for this project was, summarize all my results and inform you what my conclusion is.

For starters the purpose for my project was to test a variety of extraction kits and protocols that would determine the most efficient method for running PCR. 

I tested three different extraction kits;

• Epicentre: QuickExtract Bacteria DNA Extraction,
• Protocol Isolation and Purification of total genomic DNA
• Sigma: GenElute Bacteria Genomic DNA Kit

I tested different bacteria with each kit;

• AgroBacterium (gram-negative)
• Bacillus subtilis (gram-positive)
• Escherichia coli (gram-negative)    
• Salmonella (gram-negative)           
• Staphylococcus aureus (gram-positive)
• Staphylococcus epidermidis (gram-positive)

Bacteria	Epicentre	Sigma Extractions	Isolation and Purification of genomic DNA Extractions		PCR Results
AgroBacterium tumefaciens	Positive	Positive	Negative	Positive
Bacillus subtilis	Positive	Negative	Negative	Positive
Escherichi coli	Positive	Positive	Positive	Positive
Salmonella	Positive	Negative	Negative	Negative
Staphylococcus aureus	Positive	Negative	Negative	Positive
Staphylococcus epidermidis	Positive	Negative	Negative	Negative

 The table you see is the same table I included in my poster, this table shows which kits and protocols worked for each bacteria and the last column shows which bacteria had positive results after running PCR on them. If you can see the protocol that had the least amount of successful extractions was the Isolation and Purification of genomic DNA, while Epicentre had the most successful extractions and the Sigma worked for the gram-negative bacteria.

Project Time

Hello everyone, I hope you are all doing great! I know as the days go by we all just get busier; I am pretty sure now more than ever since our conference is only two weeks away. How exciting, I know I am both excited and anxious about it.

This is exactly what I have been focusing on this week. I must say doing the experiments and writing observations down as well as trying to figure out what went wrong or modify something for better results is interesting and fun. On the other hand it is completely different from gathering all this information and writing everything down in a formal way. This can be a bit challenging for me, but not impossible. I am now in the process of finishing my project and I am glad to say I am only two sections away from being done. Yay!

On another note I have not shared with all of you the last few PCR results that I got for a variety of primers and extractions. After I got positive results for using primers 3 and 4 from the QuickExtract Bacterial DNA Extraction Kit I tried primer 7 and my results were great! Gave me a boost in self-esteem when I thought it would never work for me. I also tried a different protocol the Sigma: GenElute Bacterial Genomic DNA Kit to extract DNA from gram-negative bacteria, these happened to be E. coli and Agrobacterium. When I ran the electrophoresis gel I had great results, I was successful in the extraction for both bacteria, however Agrobacterium DNA was way more concentrated than E. coli and I can say this by noticing how bright Agrobacterium’s’ DNA looked compare to E. coli’s’. I must say this protocol took longer than the QuickExtract Bacterial DNA Extraction protocol but I was extremely content with the results. The next step after getting positive results was running the DNA samples in the Thermal Cycler for a PCR. For these two samples I used four different primers, these primers are 3, 4, 6, and 7. I was once again satisfied with the results because the polymerase chain reaction worked for both bacteria. I must share how after running the DNA samples in the thermal cycler my results showed more concentration for the E. Coli DNA and less for the Agrobacterium DNA. I am now on the hunt to figuring out why

PCR Results for Primer 7

Ruler, E. Coli, S. Aureus, Bacillus, AgroBacterium

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PCR Time !

 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.

 

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



Primer4
E. Coli, S. Aureus, Bacillus, Agrobacterium

Primer 3
E. Coli, S. Aureus, Bacillus, Agrobacterium

Third times the charm




Figure 1
Ruler  E. Coli S Aureus, Bacillus Agrobacterium
 

Last week I worked on my DNA extractions. The bacteria I worked on were E. Coli, S. Aureus, Bacillus, and Agrobacterium. To test if my extractions were successful I ran a gel for each. I have some pictures of what I see after running the gel. I allowed the gel to run for 35 minutes at 120 volts, when that was done I placed it under UV light, and that light allows us to see if DNA is present or not. The reason we check if DNA is present this way is because to each sample I add two different dyes if any of you recall, one of them (orange dye) gives the DNA bonds higher resolution  in the agarose gel while the syber green stains the nucleic acid bonds from the DNA allowing it to absorb blue light (UV light) and emits a green color. When I place the gel under this UV light I should be able to see green light for each of my samples showing the existence of DNA including the ruler. For example in figure 1 (my first extraction), you can only see about three bacteria not including the ruler. The ruler has about 5 bright dots in its row. You can only see a big dot for E. Coli, a small dot for Bacillus, and just a line for Agrobacterium. This extraction worked for those three bacteria but failed for the S. Aureus, but none of the samples actually ran all the way down like the ruler did. This gel is 1% agarose a normal percentage to use for a gel, but in this case this gel was too concentrated and for this reason the samples did not run all the way down like they should have. If you notice the only bright bands you see at the other side of the rectangle is the rulers, not the bacteria's DNA. On figure 2 (my second extraction) though, you can see a bright light for all of them, even though you can barely see the S. Aureus band. This extraction worked for all four but even though the gel concentration was changed to .5%, the samples did not run all the way down. Figure 3 (my third extraction) though is the best extraction I have done so far. You can notice the difference in the brightness, and the length that each travelled ( almost to the ruler). By length I mean how far each sample ran to the bottom part of the rectangle.




Fgure 2
Ruler E. Coli S Aureus, Bacillus Agrobacterium
 

Figure 3
Ruler E Coli S Aureus Agrobacterium Bacillus