DNA Chips-How the genes were affected

Results from our last lab involving microarrays were some of the samples had pink in them! This symbolizes that they represented lung cancer. The samples that showed up blue meant that they appeared in both normal and cancerous cells. The fourth gene was clear, which meant that it was not present in either cell.

DNA Chips- how genes affect disease

In this next lab that we are doing in class, we will be examining how to use micorarray analysis. If a person wanted to find changes in transcription in a specific tissue, they would use this process. One microarray can contain over 30,000 "spots of DNA" where each spot represents a different gene. The first step is to make a DNA chip, that is arranged in a pattern and that represents part of a genome. The chip could then be used to analyze complementary DNAs that were made from mRNA isolated from the same tissue. The two samples of tissue are dyed and then applied to the prepared "chip". You can determine the extent of transcrition by observing how each dyed sample adheres to its complement. Once you find this out computer analysis will reveal what similarities the two samples of tissue could have.  The analysis allows scientists and doctors to find problems in certain tissues much earlier than ever before.

We will be studying six genes in this lab:
C4BPA- helps initiate part of our immune system to kill pathogens
ODC1- codes for an enzyme in the polyamine biosyntheis pathway
FGG- encoded in this gene is a part of fibrinogen, a protein found in the blood, creates blood clots
HBG1- expressed in liver, spleen, and bone marrow
SIAT9- catalyzes the formation of a protein called GM3
CYP24- catalyze many reactions involved in drug metabolism and the making of cholesterol, steroids and other lipids

CSI- We've gotcha now

In this lab we began our identification of the murderer by first putting our tube with the restriction enzymes in an ice bath. Then we collected our evidence from the crime scene, in five test tubes. We had narrowed it down to five suspects! We got a sample of each suspects DNA and placed them in test tubes, then we added 10 ul of the enzyme mix into the samples and mixed well. Next, we placed the test tubes into an incubator at 37 degrees Celsius for 45 minutes. Mr. Chugh took out our test tubes at the correct time and got them ready for when we returned the next day.  

The next day when we came back we removed the tubes from the water bath and then we gentle tapped the tubes on the table to bring the liquid to the bottom of the tubes. Next, we added 5 ul of loading dye into the separate tubes, collecting the samples in the end of each test tube. We took the agarose gel and placed it in the electrophoresis apparatus. We  checked that the wells of the agarose gels were near the black negative electrode and the bottom was near the red... But we found that our positive and negative was switched! So, we replugged the electrophoresis to correct the error. The next thing we did was using a separate tip for each sample we loaded the DNA samples into the gel and after putting the lid over the chambers we turned on the power. When we returned the next day, we could finally compare our results! We found the real murderer and solved our simulated crime scene investigation.

CSI Lab- Recognizing the Culprit

In our next lab we will be comparing band patterns produced by restriction enzyme cleavage of DNA samples. Restriction enzymes are enzymes that move down the helix until it recognizes specific sequences of base pairs that tell the enzyme to stop moving. Then the enzyme will cut or chemically separate the DNA molecules, this spot is called a restriction site. When DNA is being looked at to compare, as if on a crime scene, the way you tell the difference between two different suspects is by looking at their DNA samples. Each persons DNA is "cut" differently by the restriction enzymes, in different fragments of size and length.The DNA can be observed by using agarose gel electrophoresis. Agarose Gel Electrophoresis separates DNA fragments by size when they are put in an "agarose gel slab" and then put into a separate tube with a buffer solution. The DNA fragments are negatively charged and when the agarose gel acts as a molecular sieve where only smaller DNA fragments will travel farther than larger ones. The fragments that are the same size will band together and we will see them when we finish our lab! 

In a crime scene investigation to determine whose DNA is found, the DNA must be examined and you must  look at its nucleotide sequence. Radioactive probes are what is used to locate, identify, and compare individuals DNA. DNA can be found form many different biological materials, such as, blood, hair and body tissues. the recognition plays a large part in today's crime scene investigations. In this lab we will learn the general bases for using DNA in modern times.   

Biofuels Results!

On the first day of our lab we began our experiment by filling five cuvettes with stop solution. Then we filled both two conical tubes labeled"Enzyme Reaction"and "Control" with the substrate (cellobiose). After pipetting 500 ul of solution from the "Enzyme Reaction" tube into the first cuvette we began our timer and at intervals of one, two, four, six, and eight minutes we added the substrate to the stop solution. Immediately we noticed that the cuvettes containing the stop solution had a color change from clear to a yellowish color. This was the artificial substrate making the glucose illuminate.

 On day two, we brought in store brought mushrooms. We weighed out 1 gram of mushroom and then ground up the mushroom with a mortar and pestle, for every 1 gram of mushroom extract we added 2 ml of extraction buffer. Next, using a centrifuge, we "pelleted" the solid particles by spinning at top speed for 2 minutes. After this we labeled our cuvettes from 1 until 6.  We obtained a conical tube and pipetted 3 ml of substrate into the tube, which we labeled "button" after the type of mushroom we used. As we did the day before, we added the extract (mushroom) at 1 minute, two, four, six and eight. Surprisingly the mushroom extract made the solution in the cuvettes have the same reaction, a change of color! 

Because the reactions were the same, they pose an idea. If the reactions with a natural extract were similar to a chemical than maybe we could use other organisms in nature as substitutes for chemicals. The idea of this lab was to show us how it may be possible to come up with new ways of obtaining resources that are less harmful to the environment.  

Biofuels Lab Introduction

Biofuels are groups of fuels that are produced from a source in nature that was recently living, versus fossil fuels which are made from sources that have been dead a long time. Biofuels are being increasingly used as alternative energy sources. There are four ways to produce biofuels, through cellosic ethanol,where a breakdown of cellulose to glucose followed by a fermentation step to ethanol. The biofuel industry uses cellulases in this method. Cellulases is an enzyme that converts the cellulose in plant cells to glucose and other sugars. An enzyme is a catalyst that speeds up the process of a chemical reaction. The enzyme is very helpful because it reduces the energy needed to make the reaction occur, therefore the chemical reaction can occur much faster. The next method is the breaking down of starches and sugars followed by a fermentation step to ethanol.  The third, involving biodiesels (fuels derived from oils) are found in recycled cooking oils or in plants that produce high levels of oils that are purified and burned in diesel engines. Scientists are always trying to find new ways to create biodiesels through modifying algae, yeast, and bacteria. the last method of creating biofuels are syngas. Syngas stand for synthetic gas, it is a mixture of carbon monoxide and hydrogen gases which becomes a burning biomass. Syngas can can be burned directly or chemically converted to be used as diesel. Biofuels are important to us because they are the answer to our problems as far as global warming. If we can find natural ways to make energy we can save our environment. Although scientists are working hard, we still have a long way to go, and if we do find an alternative there are still bound to be consequences.   

In our lab we will be exploring how biofuels work, and we will work towards understanding how biofuels could change the world we live in to be more efficient. We will have multiple test tubes that will contain a strong base and then we will be adding different amounts of cellobiase (an enzyme) and P-nitrate phenol at different time increments to see if fuel is created! On Day Two we will bring in mushrooms to grind up and add to our mixtures to see a new reaction.

DNA Extraction Results

On Tuesday, September 14th our class did our DNA extraction lab. After completing all the steps in our lab, we were able to isolate the DNA in the center of our test tubes. We revealed what looked like a floating, clear, stringy substance. After letting the DNA sit for 5 minutes, we inverted the test tube and the DNA strung itself out and we were able to see the double helix shape of the genetic information! This real life encounter with the building blocks of our beings was incredible to see, and to make it all the more better we got to take our DNA home in a necklace! This lab let us see the amazing and unique shape of DNA that is located inside every single one of our cells.

Cheek Cell DNA Extraction

Our second lab of the year is designed to teach us about our DNA. In the lab we will actually extract our own DNA and put in a bottle to attach to a necklace!

DNA is present in all living things, it is the "carrier of all genetic information" and it determines everything about you. It also carries instructions to tell your cells what to do, and how to do it. In this lab our goal is to precipitate the DNA in order to be able to see it by itself, and therefore futher our understanding of this tiny thing that has quite a bit of power over our bodies. From what we know from scientists, DNA is in the form of a twisted stairway type of structure. The each "step" of the stairway is made up of bases, there are four of them named, adenine, guanine, thymine, and cytosine. Each of the bases is connected to one sugar and a phosphate group, the full grouping of a base, sugar and phosphates, create the backbone of the DNA. The bases are organized so that they each base pairs up with one other base, adenine with thymine, and cytosine with guanine. The reason for the matching of the bases are because they make messages that can be understood by genes (inside the cell), and then the genes send the information to the proteins which control the basis for the body's structure and function.  

In this lab, the first step we will be taking is we will loosen our cheek cells by chewing on our cheeks, then we will use a saline solution (0.9 % salt water) to swish around our mouths, after 30 seconds we will replace the contaminated water back into the cup in order to create a funnel into a test tube to consolidate our salt water now ridden with cheek cells. The reason for using saline solution is to neutralize the DNA and make it hydrophobic (water-hating). Next we will add a lysis buffer to break down or dissolve the cell membranes in our cells, and then we will add 100 mL of protease to break down the proteins. After those steps we gently invert our test tubes, to mix up the solution. In addition to all these chemicals, next, we will put our text tubes in a water bath of about 50 degrees celsius for ten minutes to speed up the reaction. The last thing we will do is add 5 mL of cold ethanol. After doing this procedure, we should be able to see our DNA consolidated in the center of our test tube, revealing the most important piece of genetic makeup for our body!

Yogurt Lab Discussion....

On Wednesday, June first, our class started the Yogurt Lab. We began with four test tubes...
The first had only milk in it (negative control), the second had milk and yogurt (positive control), the third had yogurt and ampicillian and the last contained E.coli.

After the first three test tubes were inoculated with a pure culture of suspected pathogen using an "inoculation loop", and the E.coli was inoculted with the E.coli pathogen, we mixed them up using the vortex, which we all had fun with! The next thing we did was to make sure everything was labeled and then we put them in the water bath at 37 degrees celsuis, in order to keep them at a constant warm temperature to maximize our results,and to see how the bacteria progressed when we returned the next morning,

Results: The next day.... We removed the test tubes from the water bath and looked at our changes. The first test tube (with only milk) had a chunky white formation at the top and a clear liquid throughout, the milk had, as a general defintion, curdled. The second test tube, with milk and yogurt had a white fluid, the third (yogurt and ampicillian) was a clearish liquid. I predict the reason that no solid was formed was because ampicillian is a beta-lactum antibiotic, which attacks the cell walls of bacteria, not allowing them to continue their growth. The last test tube had E.coli in it, this tube was the tube that by looking at it, you knew it smelled and we each bravely smelled it, determining the foul smell of rotten eggs. This test tube was a milky liquid also. After this we tested each tubes ph;
#1-ph 6
#2-ph 6
#3-ph 7
#4-ph 6
Our control test tube, containing the yogurt had a ph of 4.
 A video of our results/procedure in action is below:
http://www.youtube.com/watch?v=vVhIArPkyi0

By infecting each tube with bacteria, we found out that the "yogurtness" disease could be spread through contact with the bacteria.

Possible sources of error, a substance not being infected by the yogurt enough, a temperature issue involving the water bath, and an incorrect reading of the substances ph, could all contribute to errors in the experience.

Yogurt Lab

On September first in Mr. Chugh's Biotechnology class we will be doing a lab where we will make yogurt. In this lab we will be exploring the possibilities, along with various other objectives, that there are some types of bacteria that are good for us, unlike the common accusations of all bacteria being harmful. Bacteria are prokaryotes, meaning single-celled organisms, that are found everywhere, but only with the help of a microscope. A common presumption of people is that all bacteria is bad for us and causes diseases, but in reality there are a lot of species of bacteria that are helpful. An example is a commonly misunderstood bacteria called E.coli, it is normally presumed that E.coli is the cause of disease, but in reality we need this bacteria, because it is used to break down sugars in our bodies. In the United States it is necessary that yogurt has bacteria called Streptococcus thermophilus and Lactobacillus bulgaricus in order for it to be classified as yogurt.  

In order to further our understanding about bacteria, we will be testing a German scientist named Koch's postulates, to do this we will be studying pathogens by seperating them from a substance the pathogen has already infected, then we will use the same pathogen to infect other substances and then compare our new results to the originals. Our results will tell us whether or not the bacteria we are using causes a particular disease, or in this case we will be finding out whether we can recreate our yogurt identically both times we do the experiment. In the lab, we will be creating yogurt by scalding milk, bringing it to 80 degrees celsius, then we will allow the milk to cool off and we will add "yogurt bacteria" and mix. When we return to class the next day, the bacteria will hopefully have done its job by soldifying the milk and creating a solid, creamy solution. Yogurt! This lab gives us the opportunity to become comfortable and familiarise ourselves with the happenings of this class, and proper technique while doing a lab.