CSI: Acalanes. DNA Fingerprinting

      Restriction Enzymes are enzymes that are used by bacteria and other cells to route virus attacks. When a virus attacks a cell it tries to inject its own DNA into the cell and use the cell to produce more viruses. Restriction enzymes in the cell will try to stop this by "cutting" the intruding DNA at certain very specific DNA sequences. In the real world restriction enzymes are used in DNA fingerprinting. DNA fingerprinting can be useful in criminal cases to determine who did what or touched what. DNA fingerprinting is a semi complicated procedure. First one must get the DNA from some type of bodily fluid or other material that has DNA in it. Then one must extract the DNA and introduce the restriction enzymes to it. At this point it is better to put the DNA+Restriction enymes into a warm water bath of about 37 degrees celcius. This temperature will allow the enzymes to work with maximum efficency since it matches the temperature of the body which is were the enzymes came from (E.Coli in large intestine). After letting it sit for a while you can take the resulting material and put it into agarose gel. You can then run an electrical current through the gel and since DNA is negativly charged it will move towards the positive end of the agarose gel. After letting it run for a period of time you will have lines of DNA at different levels in the agarose gel since certain pieces of DNA that are smaller will move farther than other pieces. You can than compare this result to another agarose gel that has other DNA in it and they will be different because everyones DNA is different and so was then cut at different areas resulting in different piece sizes which would then look different on the agarose gel.
      In our lab we are trying to match the DNA of one of the suspects to the DNA from the crime scene using DNA fingerprinting. My hypothesis is that if the DNA fingerprinting (agarose gel electrophoresis) of one of the suspects matches the DNA at the crime scene it then means that they did it.
      The result of the lab was that the DNA from suspect 3 matched the DNA from the crimes scene. This means that suspect 3 commited the crime since their DNA was there. Errors could have been made when we injecting the DNA into the "wells" in the agarose gel since it was very hard to do and required a steady hand. If we had mess up when injecting the DNA then it is possible that the different DNAs could have mixed and messed up the results.






DNA FINGERPRINTING



Different lines of smaller and larger pieces



Biofuels: The Way of the Future

     Enzymes speed up the rate of chemical reactions. They are proteins that are used by organisms in order to perform some sort of chemical reaction in the environment which that organism lives. An example of an enzyme is the enzyme Sucrase which breaks down the sugar Sucrose. In this lab we want to create ethanol from plant matter. This is also being done in the real world in order to find alternative fuels that we can run machines on instead of petroleum because ethanol can be burned instead of petroleum. In order to create ethanol from plant matter we have to follow certain steps. First be have to pretreat the plant matter in order to remove non-cellulose molecules such as lignin which block the enzymatic activity of cellulases which break down cellulose into glucose which is what we want. After we remove all of the “enzyme blockers” we must then add cellulases which are enzymes that break down the cellulose into glucose. In order to detect if the enzymes are actually working we will use a colorimetric assay using p-nitrophenyl glucopyranoside which will change into a yellowish color if it is being broken down by the enzymes. Once we have glucose left we must then ferment the glucose using microbial fermentation. Microbial fermentation is using microbes such as yeast in order to break down sugars. This step will change the glucose into ethanol. Ethanol then can be burned as fuel instead of petroleum. All of the above steps and results were exactly the same for the mushroom extract portion of the lab except that instead of using cellobiase we used the enzyme in mushroom extract.
      Before performing the lab we made the hypothesis that the stop solution liquid would get more and more yellow as time went on because the enzymes would be breaking down more and more substrate. Our hypothesis turned out to be correct and as we put the enzyme reaction liquid in the stop solution at different times we realized that the more time was given to the enzyme, the yellower the stop solution would turn. We got the same results for both the the enzymes cellobiase and the mushroom extract. Many things could have gone wrong with the experiment. We could have forgotten to add the enzyme reaction to the stop solution at the right times or we could have added too much or too little of a certain material which could have messed up our results.

    

DNA Precipitation: The Human Wonder

       DNA is the genetic code our body that is located in each cell in our body and decides everything about us. DNA is a chain of nucleotides with the bases Adinine, Guanine, Cytosine, and Thinine. DNA is the "manual book" for our cells. It tells our cells how to make certain proteins. DNA does this by sending the code to ribosomes which then make the proteins based the code. DNA precipitation is a method used to concentrate/isolate DNA from a cell. To achieve this we have to break down the cell membrane and the nuclear membrane. Then we have to add certain ingrediants to be able to actually see the DNA with the naked eye. This is used in the real world as well. Researchers can use this method to isolate DNA and then take samples of it and learn more about it. They can use the info gained to test for certain traits and dieseases. In order to make this happen we have to add certain ingrediants and do certain things. First we have to loosen our cheek cells and put it in saline which is isotonic and the same salt concentration as our bodies. Then we add the lysis buffer which breaks down the cellular and nuclear membrane. Then protease which breaks down the DNase in our cells (kills DNA (enzyme for DNA) )  and also breaks down the histone proteins which the DNA is wrapped around. We then put it into a hot bath to speed up the reaction and we add salt which neutralizes the negative charge of DNA. Lastly we add cold ethanol which helps precipitate the DNA.
      In my DNA lab I went through all the steps and got an amazing result. Once I added the ethanol I saw these thin strands of a jello like substance appear. It was really cool and I appreciated the awesomness of DNA after that. Some errors might have occured. We coulded have added to much of a substance like ethanol or salt and that could have affected how much DNA we ended up with after that . Overall I really liked the lab and had a good time doing it.




DNA



Precipitated DNA

Yogurt and bacteria for dummies

       In the 1800s people sickened and died for no apparent cause. Koch's postulates were invented by Robert Koch in order to prove that a certain microbe caused a certain disease. Bacteria are small microbes that are all around us. They are the the most successful form of life on the great planet Earth. Bacteria have no separate place for the nucleus like eukaryote do. Most bacteria are harmless and do not harm humans in any way. There are a small amount of bacteria that can harm you. There are also some bacteria that are actually good for humans and their digestive systems. These bacteria are called probiotics and can be found in yogurt and other foods. Bacteria that form yogurt are anaerobes and they use enzymes to break down milk sugar (lactose) into lactic acid.
      The purpose of this lab is to prove all of Koch's postulates especially postulate 3. Postulate 3 is: Inoculate healthy individual with pure culture of suspected pathogen. The oobjective of the lab is to use yogurt, ampicillin, and e.coli to prove this point. The technique we are using to prove postulate 3 is used all around the world in different ways to create yogurt from milk. Koch's postulates are also used in labs to isolate unknown pathogens.
       In the experiment we have 4 tubes of milk. We add ampicillin to the Yogurt + amp tube. Then we add yogurt to the yogurt + amp tube and another just milk tube. We are doing this to show that the pathogen is a bacteria because ampicillin kills bacteria so if nothing happens to that tube and the other tube becomes yogurt we know it was the bacteria. After all of this we then add e.coli to a tube of milk. We do this to show that introducing bacteria itself can cause the change to yogurt therefore isolating the pathogen.
       When we recieved our tubes back we came up with some expected and unexpected results. The negative tube that was filled with just milk had stayed in liquid for which was to be expected. The E.coli tube had also stayed in liquid form which had also been expected. The tube which had Yogurt + amp had changed into yogurt which wasn't supposed to happen. Also the tube that contained just milk with yogurt had stayed liquidy. I think this occured because of human error. I think that my group miss labeled the tubes with yogurt+amp and yogurt therefore switching them and explaing the unexpected results.

E.Coli

Yogurt

VIDEO:
http://www.youtube.com/watch?v=fKG-6jEwh_0