Human Mitochondrial

The mitochondrial (mt) genome which contains only 37 genes, is used for the process of the production of energy and its storage in ATP. In the "Human Mitochondrial DNA Kit" background, it said that there was strong evidence that mitchondria once existed as a free living bacteria, which were taken up by primitive ancestors of eukaryotic cells. The MT genome has some bacteria like feature, is also a circular molecule and very few noncoding sequences (introns) interrupt mt gene. In 1981, the entire DNA sequence of the mt genome was determined. In the mt genome, there is a region of noncoding nucleotides (1,200) in which it contains signals that control replication of the chromosome and the transcription. So this DNA sequence is called the hypervariable because it accumulates mutations at approximately 10 times the rate of nuclear DNA, and because of this, ther is a unique pattern of single nucleotide polymorphyism, which could be inherited through generations. Also in the 1980's, Alan Wilson of UC Berkeley, used mtDNA polymorphisms to create a family tree, that showed the ancestral relationships of modern people. From this they were able to conclude that all modern humans arose from Africa about 200,000 years ago. Today mt DNA has been used to identify the remains of unkown soldiers in Vietnam, also determine the reamins of the Romanov royal family and the relationship of Neandertal remains to modern humans
Directions:
Step 1. First we labeled one screw cap tube containing 200 hl of InstaGene matrix and then rinsed our mouth with saline solution for 30 secounds. (chewed inside of our gums to loosen cheek cells.).
Step 2. Then we placed this saline solution that we spit out into the cap tube containing the InstaGene and then we centrifuged it for about 2 minutes.(After it was centrifuged, there was a white bead of whitish cells at the bottom of the tube.)
Step 3. Then after centrifuging, we poured out the extra saline solution out of the tube, so then we only had the pellet of white cells at the bottom of the tube.
Step 4. Then we vortexed the tube so there would be no clumps of cells remaining and to resuspend the pellet of cells.
Step 5. Then we transfered the resuspended cells, into the screwcap tube containing InstaGene.
Step 6. Then we vortexed the tube and then placed the tube into a water bath (56 degrees Celcius) for 10 minutes.
Step 7. After we placed the tube in the vortex again and then put it into a boiling water bath (100 degrees Celcius) for 5 minutes.
Step 8. Then the next day we placed the mixture into a PCR tube and also added some yellow master mix. Then we placed it into the thermal cycler for 40 cycles.
Step 8. Finally, we added loading dye to our PCR tubes and then placed our mixtures into the wells of the agrose gel and then turned on the electrophoresis apparatus for about 10 minutes.
Results: Our results showed that we were all negative for the disease, but it could possibly be wrong because we could have messed up when loading the mixtures into the wells of the agrose gel. Since our results were very faint and very hard to see if they really were negative for the disease. I also think that my tube might have not had enough cheel cells because when I tried transfering the cheek cell bead to another tube, it sort of feel apart.So this may have caused me to transfer more of the saline solution rather than my cells.

DNA Testing

Background: In todays modern world, we use Molecular biology, is the study of of genes and other molecular structures, that regulate the flow of genetic information from generation to generation. Also Biotechnology uses this information to help solve human problems, by taking this information and testing it through DNA tests, just like our very own lab. In our our lab we are doing a DNA test, to find out whether or not certian people in class have a specific gene that we are tryign to target. This DNA testing is used for all sorts of things, but it is most commonly used for helping people find out if they have or carry a certain disease like Huntington disease. In order to do this lab we must use PCR, which is a technique that takes a trace of DNA and produces a large amount of it. This technique today is widely used in biotechnology and it even helepd transfrom molecular biology into a multidisciplinary research field within five years of it being made. PCR has also had a profound impact on four main areas of genetic research: gene mapping, gene cloning, DNA sequencing and gene detection.
Directions:
Step 1. First we labeled one screw cap tube containing 200 hl of InstaGene matrix and then rinsed our mouth with saline solution for 30 secounds. (chewed inside of our gums to loosen cheek cells.).
Step 2. Then we placed this saline solution that we spit out into the cap tube containing the InstaGene and then we centrifuged it for about 2 minutes.(After it was centrifuged, there was a white bead of whitish cells at the bottom of the tube.)
Step 3. Then after centrifuging, we poured out the extra saline solution out of the tube, so then we only had the pellet of white cells at the bottom of the tube.
Step 4. Then we vortexed the tube so there would be no clumps of cells remaining and to resuspend the pellet of cells.
Step 5. Then we transfered the resuspended cells, into the screwcap tube containing InstaGene.
Step 6. Then we vortexed the tube and then placed the tube into a water bath (56 degrees Celcius) for 10 minutes.
Step 7. After we placed the tube in the vortex again and then put it into a boiling water bath (100 degrees Celcius) for 5 minutes.
Step 8. Then the next day we placed the mixture into a PCR tube and also added some yellow master mix. Then we placed it into the thermal cycler for 40 cycles.
Step 8. Finally, we added loading dye to our PCR tubes and then placed our mixtures into the wells of the agrose gel and then turned on the electrophoresis apparatus for about 10 minutes.
Results: Our results showed that we were all negative for the disease, but it could possibly be wrong because we could have messed up when loading the mixtures into the wells of the agrose gel. Since our results were very faint and very hard to see if they really were negative for the disease. I also think that my tube might have not had enough cheel cells because when I tried transfering the cheek cell bead to another tube, it sort of feel apart.So this may have caused me to transfer more of the saline solution rather than my cells.

Genetically Modified Plants

      Genetically modified products today, are widely used in the U.S., in order to produce a better food product that can withstand harsh environments and other factors such as insects. Proponents of GMOs, believe that these plants hold the solution to to help put a stop to overusing pesticides and herbicides, that are causing grave health conditions to us and problems to our environment. Farmers have been genetically modifying their crops for centuries through crop breeding, in order to encourage specific traits, like allowing a plant to bear more fruit. But today, there is the option of selecting specific genes from anything, not just from another plant, but also from organisms. Because of this new technology farmers today, are able to produce plants that have delayed fruit ripening , resistance against fungi and even increase crop yield. Though many people today, believe that GM plants are bad because the plants are created naturally through evolution and many believe it could cause health problems to humans and create super resistant bugs and weeds. Today there are two methods of identifying if a crop is a GM plant, one method is using an enzyme called immunosorbent assay, which identifies proteins. So this enzyme, basically identifies specific proteins produced by a GM plant (can only be used on fresh produce.). Another way to test for a GM plant, is using polymerase chain reaction (PCR), which identifies sequences of DNA that have been inserted into a GM plant. In our lab, we are going to test our two foods that we brought in from home, to see if it is a GMO food/plant.

 Procedure:

Day One: First we weighed out .5-2 g of certified non-GMO food and our food that we brought in, and put it into a mortar. After, we added 5 ml of distilled water to every gram of food and then grinded it with a pestle for two minutes. Then we pipeted 50 ul of the mixture into a screwcap tube containing 500 ul of InstaGene. After this we placed our test food tubes into a 95 degree Celcius water bath for 5 minutes, then placed the tubes into a refrigerator, until the next day.

Day Two: On day two we numbered our PCR tubes 1-6, corresponding to the tubes content. The even numbered tubes got the red master mix (20 ul of master mix to each tube.), and the odd numbered tubes got the green master mix. Then we placed the tubes into an ice bath and put in master mix according to each tube, and then we placed them into a thermal cycler.

Day Three: On day three we used electrophoresis (which we placed each test tube mixture into a specific well), and then after this we were able to determine if our test foods we brought in were GMOs.



Results: I predicted that our test food, which was an orange, wouldn't be a GMO because it was grown in one of our backyards, but we found out that it was actually a GMO.  Our results showed that both of our test foods tested positive as a GMO product. Our lab could have possibly flawed though because when we were inserting our mixtures into the wells in the agrose gel, one of the mixtures in a well sort of spilled out alittle.

pGLO Lab

In this lab we are using the technique called genetic transformation, in order for us to create a bacteria that glows using a gene that codes for green fluorescent protein. Genetic transformation is used, by inserting  a gene of interest from another organism into another, in order to change an organisms genetic makeup. Today, genetic transformation is used in biotechnology in many ways, some uses of this technique are to create perfect fruits and vegetables. Today in our lab, we are using a green flourecent protien that comes from jelly fish in the dark waters of the ocean, and we are going to take this gene and insert it into a plasmid and then insert it into our bacteria. (A plasmid is a circular DNA molecule capable of self replicating itself and also able to carry genes.)

The DNA Chips

 Today, scientists use a process called microarray to find differences in gene expressions, since it allows scientists to see a specific gene in a cell. Which makes it alot easier to find these differences because you don't have to look at the entire genome to find what your looking for.  Definition: DNA microarray is thousands of microscopic spots of DNA oligonucleotides, which have specific probes on them. Scientists today, use these microarrays to find specific gene sequences that cause human health problems such as skin cancer, breat cancer and etc.
Steps for the Microarray:
1. Isolate DNA.
2. Pelimanary chains reactions make more genes.
3. Then a robot spots individual gene sequences into specific addresses onto a DNA chip
4. Then the mRNA from the tissue of interest is dyed red and also the mRNA from a second tissue of interest is dyed green.
5. Finally these mRNAs are put on the chip and analyzed.
For our Microarray lab we followed these same procedures to determine which six genes we had ( ODC1, C4BPA,FGG,HBG1,CYP24 and SIAT9), caused lung cancer, from the two samples we have from a cancerous cell and a healthy cell.

CSI: Crime Scene Investigation Lafayette

Background: Scientists today, use a bacterial defense mechanism called the restriction enzyme. A restriction enzyme is basically like a "restriction scissors" that make cuts at certain areas in a sequence of base pairs that are recognized by the enzyme. This enzyme has derived from viruses that eject their DNA into a cell, and use the restriction enzyme, as a bacterial defense. A restriction enzyme, basically slides down a DNA molecule, until it starts to recognize a specific sequence, this then causes the enzyme to stop sliding and cut the strand off that is recognized by the restriction enzyme. Once this happens scientists take this specific strand and place it into a process called agarose gel electrophoresis, which separates the strand so it can then be observed. This process is done, by placing the DNA fragments into an agarose gel slab, which is placed into a chamber filled with conductive buffer solution, then a direct current is passed between wire electrodes at each end of the chamber. Since DNA fragments are negatively charged, they are going to move towards the positive poles in the electric field. Once this happens, the agarose gel acts like a "molecular sieve" which smaller DNA fragments move easier than larger fragments. Some time after, the smaller fragments will have traveled further than the larger pieces, creating single bands of DNA at certain places, which are then stained so the bands can then be seen. This is just like DNA fingerprinting from crime scenes, but they use radioactive probes that are specific to certain nucleotide sequences and then compare the DNA of different people.

Agarose Gel Electrophoresis

Purpose: Our purpose for this lab is to figure out who committed the crime by using a forensic DNA
fingerprinting kit.

Lab instructions: 1. First we labeled micro test tubes CS (crime scene), S1 (suspect one), S2 (suspect two), S3 (suspect three), S4 (suspect four), S5 (suspect 5).
2. Then we took a DNA sample form each stock tube and placed the specific DNA samples in the corresponding tubes (using a 10 pipet).
3. Then we used a centrifuge to mix the components together.
4. Next we placed our tubes in a foam micro tube and incubated them overnight in 37 C water.
5. Then we added 5 hl of loading dye in each tube.
6. Then we added each sample in its specific well in the agarose gel, while it was in the electrophoresis apparatus with 275 ml of buffer solution.
8. After that, we turned on the electrophoresis chamber for 3 minutes and then turned off the apparatus.
9. Next, Mr. Chugh stained our agarose gel with 120 ml of 1x Fast Blast DNA staining it overnight.
10. The next day we saw our end results of our lab.

Observations and Results: Once our agrose gel was complete I could easily see deep blue strands of DNA in certain places on the gel. In our end results, we came to a conclusion that suspect 3 or Chloe had to have done the because, the DNA strands from suspect 3 (Chloe), was closely related to the one found at the crime scene. Though we were sure that our results were correct, we could have accidentally not put enough of the DNA in well # 3, since the DNA strands in 3 were very faint and hard to see where the location of the strands were in the agarose gel.

Enzymes and Biofuels

Background info:
 General information about Enzymes: Enzymes help speed up the process of chemical reactions, and are able to work again and again, converting reactants into products, since they do not react with substrat.(Substrat is the reactant in an enzyme-catalyzed reaction.)
Cellulase: Cellulase is the structural polysaccharide found in the cell walls of plants, that is only contained in plants because it's the source of sugars for the plant. Though animals cannot produce cellulose, some animals that eat plants, have a bacteria that produces cellulose. For instance cows produce cellulose because they have a bacteria called Bacteriodes succinogenes, that produces cellulose for them.
Cellulose practical uses: Cellulose today, is used in the bio fuel industry to create ethanol that can help run cars, planes and more. Cellulose is converted into ethanol by extracting the cellulose from a plant's cell wall then converting it into a sugar, and finally the product is changed to ethanol through microbial fermentation.

Purpose: Our purpose for this experiment is to gain more research about the reaction rate in the presence of an enzyme.

Lab instructions:
First we label five curevttes E1-E5, then we put stop solution in all the curvettes.  Because we are adding the stop solution, it will dentaure the enzyme reaction, which will cause p- nitrophenal to turn yellow. Since this solution will turn yellow, it will show how much of a reaction is happening. After this, we will then add substrate to both the enzyme reaction tube and the controlled tube. Then, we will add a buffer to the controlled test tube and then put this solution in the curvette labeled E1. After, we then add some of the enzyme reaction solution to the curvette and then start our timers. After this, we will then add more enzyme reaction to each curvette at set times. On day two of the lab, we used a mushroom as our enzyme instead, by extracting the enzyme in the mushroom with the centrifuge.

Hypothesis and Results: I think the curvettes that have had the longest length in contact with the reaction solution, will be a more darker yellow, while the ones that weren't in contact with the reaction solution, will have a light yellow color. My hypothesis was correct, as we put the solution in the curvette and let it set for a longer time, the more yellow it would turn,which meant more of the product was being produced. Though, we believe our end results were very accurate it, we could have had one mistake, which could have been using dirty curvettes that weren't thoroughly washed from the last period. We also learned that the reaction of the enzymes would soon end because the enzymes would lose recources to make more product, since we saw this in the mushroom lab.