Cancerous "CHIPS" - not the salt kind......

Background
The micro array chips is the most powerful new research tool that makes the viewing of thousands of genes expressed in cells. It is bess used for looking for patterns or changes in transcription in cells which makes the study of normal and abnormal of the cells function easier.

A singel micro array can contain more than 30,000 spots of DNa, each is representing a different genes in the organism. This chip is used to study the expression of six different genes in normal lung cells and lung cancer cells:

Gene 1
The protien that codes for this gene helps start our immune system to kill the pathogens

Gene 2
The gene helps code for an enzyme catalyzes the conversion of omithine to putrescine

Gene 3
Codes for a part of fibrinogen, a protien found in blood that helps in the forming of blood clots

Gene 4
One of the two y-globulin genes

Gene 5
Plays the role in inducing cell differentation, controlling cell cell growth

Gene 6
catalyze many of the reactions involved in drug metabolism and maing cholesterol, steroids, ande other lipids.


Procedure:

Take the micro array slide and useing a pippet spot on the 6 different gene sequences onto the glass slide
carefully add the 20mL of hybridization solution to each spot (Dont allow the dropper to touch the spot)
Record the results by weiting a discription of the color of each spot in your results.
CLEAN UP

Results/Observations:

#1 and #5 pink
transcribed from the DNA to mRNA-> cDNA (colored)

#3,#4,#6 no pink dye showed up

#4 a non protien coating gene (could be found prodice hemoglobin)

#2 is expressed in both which means no real change from normal to cancerous cells

#1 and #5 only expressed in cancerous cells (#1,#3#5.#6 all in cancer)


Discussion:

a) even though there wasnt a real way to hypothesize the out come the results were not as what we would have thought because we havent ever done DNA mirco arrays before.

b) When we did the color of one of the spots we didnt realize that it changed purple at the beginning so over time it turned blue.

CSI: The Acalanes Happening

Introduction:

DNA profiling or "fingerprinting" to analyse evidence in the law enforcement cases and other applications. Restriction Fragment Length Polymorphism has been theworkhorseof foreignsic DNA profiling for many years. Alec Jefferies in 1985, RFLP analysis provides a unique banding pattern of an individual's DNA sequence. DNA fragments are negativly charges and are drawn toward the positive pole (anode). Over a period of time, smaller DNA fragments travel farther than larger ones. These fragments of DNA in the gel will be stained and seen to provbide evidence to DNA matching. A quick analogy the desks and chairs have been pushed together. A individual student weaves through the desks no problem compared to where the strand of 4 students have a bigger difficulty taking more time.
The sililarities and the differences in DNA are used to show the specific nucleotide sequence and are shown in the gel when stained (see in pics). A radioactive complementary DNA probe can be recognized and binded; This probe can be described as "radio active lag" thaty will bind to a single stranded DNA fragment and produce a band in the gel. This brings together the lesson of "DNA fingerprinting" where the strands in the radiolabeled bands in the gel are used by determining the size of the DNA fragments in each band. Each person has a special bandings that will reflect the variations in the indivivuals' DNA.
These DNA evidence that is needed for DNA fingerprinting cna be obtained from any biological material and even dried material such as blood stains or mummified tissue.
Restriction Digestion of DNA
Restriction enzymes are the "chermical scissors" of the molecular biologist and the enzyme recognized a particular recognition sequence of the segment of DNA.
Making DNA Visible
Dna is colorless so the DNA fragments in the gel cannot be seen during electrophoresis which is a loading buffer containing two bluish dyes that is added to the DNA samples. The lodaing dyes doesnot stain the DNA itself but makes it easier to load the samples and monitor the progress of the DNA electrophoresis. The dye will "run towards red" just like the DNA fragments. The "faster" dye with the DNA will travel through the agarose gel and the slower ones will travel as well forming the now invisible streaks in the gel.
Reliability of DNA Evidence
In general one can assume that any two humans are 99.9% identical DNA sequence thus differing by only 1 bp in 1,000.
Alternative DNA Fingerprinting Scenarios
DNA finger printing and profiling also DNA typing are all names for the same process that shows relatedness of humans, pants or animals. Some of these that could be used for is Food idenification, Accused and convicted felons set free because of DNA typing, Identifyling human remains, Determining relatedness of humans, Studying relatedness amone ancient peoples, DNA testing families, Idenifying organisms that cause disease, Identifying birth parents, Proving paternity,Determining effectiveness of bone marrow transplants, Proving relatedness of immigrants, Confirming relatedness among animals, and DNA testing of plant material puts murderer at the scene.

Procedure:

put the tube containing the restriction enzyme on ice
label the tubes according to the instructions:
green CS
blue   S1
orange S2
violet  S3
red    S4
yellow S5
label the tubes with name, date, period, table etc and put in tube holder
using the fresh tip for each sample pipet 10ml of each DNA sample from the stock tubes to transfer to the colored tubes
put 10micro liters into the bottom of the tubes to make sure not to suck it up
put the caps tightly on the the tubes and put onto a centrifuge and pulsate to collect all the liquid at the bottom
place the tubes in a water bath incubator for 45mins or overnight
when incubation period is done put in the refrigerator until the next lab period

DAY 2

remove the digested DNA samples from the refrigerator
use the centrifuge to bring all the liquid to the bottom of the tube
using a different tip for each sample put some loading dye into each tube and gently mix by contracting and retracting the pippet
remove the agarose gel from the refrigerator
place the agarose gel in the electrophoresis apparatus. fill with 1x TAE buffer to cover the gel
remember "run to red"
using a seperate tip for each tube put the samples into the agarose gel
put the lid on the elctrophoresis apparatus and run the power on 200 V for 12 mins

Visualization of DNA Fragments
when the run is complete turn off the power and remove the top of the chamber and remove the gel to put inot the box
Overnight Staining
add  enough Fast Blast DNA stain to fill the top of the agarose gel and let stain overnight and put on the rocking table to make sure even staining.
drain the gel to make sure the gel is dry to be able to look at the bands the next day

Results and Observations:

Our group came to the consensis that Katie Records killed ME!!!! Also when the pippeting was done one of the time we may have contaminated one of the tubes but for some reason it didnt effect the results of the DNA finger printing.


Discussion:

a) I really had to idea who would have done it but I thought Mr.Chugh would pull a fast one and make it a crazy suicude and have me frame somebody. Also i had no idea how well this actually work in the forensic aspect of crime solving in the fields.

b) There could have been contamination in one of the tubes but it seemed to not affect any of the results thankfully.

Biofuels of Mankind

Intro:

Enzymes speed up the reaction process and the properties of the active site is important becuase it is where the reactant(s) binds . The reactant in an enzyme-catalyzed reaction is called the substrate and it fits the active site because the amino acids facing the active site attract. The enzyme speeds up the chemical reaction by putting it in such a way to stablize the reaction.

All the concentration of all of the molecules involved in the reaction affects the reaction rate, for example the more of the enzyme the faster the reaction. Increaseing the reaction until the point the enzyme present is saturated with the substrate, which brings the example of "increasing the amount of raw materials will increase production.

Wich leads into callulose, found in the cell walls of plants, is the source of sugar to organisms that produce cellulases. Cellulases catayze the creakdown of cellulose to glucose. Humans and other animals do not produce celluases. Some plant eating animals are hosts to other organisms that possesses the enzymes for example: termites that have protozoan in their gut that breaks down wood.

The biofuel industry uses cellulases to convert the cellulose in plant cell walls into sugar which convert into ethanol by microbial fermentation. The ethanol tcan be use in certain engines or in comboination with gasoline to power cars, trucks and airplane engines. For celluosic ethanol production, lignins must be removed because they inhibit enzymatic activity of cellulases.

Procedure:

DAY 1
get the 15ml conical tubes labeled: stop solution, 1.5mM Substrate, Enzyme, Buffer
label 5 cuvettes E1-E5
label the two remaining cuvettes "Start" and "End"
use DPTP, to pipet solution into each cuvette
label empty 15ml conical tube "Enzyme Reaction" and the other "Control"
use clean DPTP \, put 2ml of 1.5mM substrate into the 15ml conial tube labeled "Enzyme Reaction". Then pipet 1ml of 1.5mM substrate into conical tube labeled "Control"
label one DPTP "E" for enzyme and the other "C" for control
use DPTP labeled "C" to puit 500 of buffer into the 15ml conical tube labeled control and mix
once mixed add it to the cuvette labeld "start"
pipet 1ml of enzyme into 15ml conical tube labeled "Enzyme Reaction" THEN START THE TIMER
remove 500 of the solution from the "Enzyme Reaction" tube and add it to the right labeled cuvette with stop soluation
after all the enzyme samples have been taken, use the DPTP labeled "C" to remove 500 from the "Control" reaction tube and add it to the cuvette "End"

DAY 2
Take the mushroom and take 1 gram of it
grind it in the mortar and pestle to make a slurry
add 2ml of mushroom
put the tube onto a seperator go-round
then label the cuvettes labeled 1-6
use DPTP, to pipet solution into each cuvette
label empty 15ml conical tube "Enzyme Reaction" and the other "Control"
use clean DPTP \, put 2ml of 1.5mM substrate into the 15ml conial tube labeled "Enzyme Reaction". Then pipet 1ml of 1.5mM substrate into conical tube labeled "Control"
label one DPTP "E" for enzyme and the other "C" for control
use DPTP labeled "C" to puit 500 of buffer into the 15ml conical tube labeled control and mix
once mixed add it to the cuvette labeld "start"
pipet 1ml of enzyme into 15ml conical tube labeled "Enzyme Reaction" THEN START THE TIMER
remove 500 of the solution from the "Enzyme Reaction" tube and add it to the right labeled cuvette with stop soluation
after all the enzyme samples have been taken, use the DPTP labeled "C" to remove 500 from the "Control" reaction tube and add it to the cuvette "End"


Results and Observations:

When adding the stop solution it caused the cuvettes to notg kepp getting yellower then it could have been
grinding the mushroom into the slurry didnt look like it would work as the enzyme


Discussion:
a) personally the hypothesis of grinding the mushroom I thought it wouldnt work at well as using pure enzyme from DAY 1

b) When grinding the mushroom slurry there could be errors of not grinding it enough and not releasing the enzyme in the slurry.

DNA that necklace

Intro:
a) DNA is in all living things including bacteria. plants, animals and most cell types. The carrier of the genetic information consisting of hair, skin, and eye color, facial features and everything that hs to do with the appearance of people. It also carries info for the cells to do their normal function and are known as the "blueprints". This consists of your father's and your mother's DNA.
DNA looks the same in everything but seeing your DNA is cool, knowing that it is the info that constructs you. The extraction is simple and is done by scientists all the time from different organisms.
b) Everyday scientists use the DNA to make new discoveries through the code embedded in the strands. These new discoveries include cures for diseases tat could be the answer to the people suffering.
c) 15ml tube, saline solution, plastic cup, plastic pipette, cold alcohol.
d) The DNA when extracting will appear in a cloudy bunch on the top of the alcohol.

Procedure:
Get a 15 ml tube and put 3ml of saline solution in the tube.
gently chew the inside of the4 cheeks
take the solution and swish it in the mouth for 30 sec
spit the solution back into the cup and then put back into tube
get the tube and put 2ml of lysis buffer in it
put the cap on the tube and invert the tube 5 times
add 100 ml of protease
put cap back on and invert
place the tube in the 50 degree celsius bath for 10 mins
add the 10 ml of cold alcohol
let sit for 5 mins
after invert the tube to help the DNA gather
get the plastic pipette and extract the DNA

Results/Observations:
After adding the lysis buffer the liquid seemed to thicken and when we were done the DNA clouded up and gathered up into a ball.

Disussion:
a) The hypothesis we all got was as we thought would happen, we thought the DNA would form when the alcohol was put in the tube.
b) When we got the DNA there seemed to not have as much DNA as i thought there was going to be. I may have needed to chew on my cheeks a little more or better to have more cheek cells.

BACT to the yogurt

Intro:
During the 1800's the the fear of microbial spread diseases because of the people that died were dying for no reason. Some of the early scientists which included a man that his name was Koch, a german scientist that wanted to prove the reason of is microbial deaths. In which he formed the Koch postulates that is described as a unethical and dangerous to experiential on humans so they would use animals to study further in e fields of humans disease.

Milk will turn into yogurt and tic ken over time after the bacteria has been introduced also put in one phrase the "yogurt disease". But using Koch's postulate it will prove that the "yogurtness disease" is not in the yogurt. This experient will tell if the micro bacteria is the cause if the yogurtness disease. While yogurt is getting the bad rap of causeing yogurtness disease the store bought yogurt is perfectly healthy and is very healthy. Only a flew small amount of have caused disease in humans but, the healthy bacteria outwieghs the bad and is good for digestion and makes you healthier.

Ampicillin may casue allergic reactions that could infect the body from the eyes, respritory system, and the skin. If contact is made in the eyes rinse out right after with water and get medical attention. Wearing protective can prevent these situations from happening. Ampicillin is a antibiotic and people with allergies should aviod the contact with the antibotic which could include penicillin.

Bacteria, the most important life form on Earth has more bacteria than species on the Earth. Bacteria can be found anywhere from: soil, durt, water, animals, in and on plants, in and on humans and also underground. Being classifed as prokaryotes, single-celled that are too small to be seen without a microscope. Unlike eukaryotes  there is not seperate place that the bacteria stores their DNA. Some characteristics also include the can grow tail to use for movement, sometimes they form chains (some will be seen in the lab). But when we think of bacteria we unfortunatly think of disease but there arer only a tiny amount of bacteria that does so and has been part of our history on Earth. The use of antibiotics has lowered the possiblility of those bacteria diseases but has left us on  the track of contracting the old diseases. In milk we can find tons of bacteria because milk is a perfect place to find bacteria because it is the cause of spiolage. To prevent this from happening we heat up the milk and rapidly cool it to keep the milk from spoiling longer by killing most of the spoil milk causing bacteria. Anton Van Leeuwenhoek of the Netherlands first saw bacteria in 1676 and called them tiny animals. In 1875 Robert Koch was actually able to prove the germ theory which lead him to set the rules called the Koch's postulates.

The shapes and variety of bacteria are consisted of spherical, rod-shaped, and spiral. Bacteria divide and multiply and over time can become millions and even billions but these form visible colonies. The different types of cell walls classify a differently to each type of bacteria. Being a living organism like human re quire food, which could include sugars to use for their daily fuction or even some bacteria need oxygen from the air to grow. The bacteria found in yogurt are using milk sugar this reaction will cause the milk to thivken or curdle to for the yogurt that we buy in the stores.

Koch's postulaes were a big part of the study of bacteria and provided evidence that they didnt just appear out of nowhere also serve a major role in fermantation. To demonstrate the postulae you must: describe the symptoms shown, isolate and identify the pathogen, use the pure culture to infect the new material, and isolateand identify the organism. The benfits of bacteria are major in the fermentation of foods such as kimchi, sour cream, pickles, cheese and yogurt. Also the other benefits of bacteria include the antibiotics which came from the study of the bacteria and formed the drugs today that fight the bad bacteria.

The Sterile Technique is the aviodance of contamination which can be found everywhere and mostly on ur hands and lab benches. It would be helpful to you and others to clean up the banches when you are done with use.

b) The purpose of the experiment is to show you how to make yogurt and be able to see how bacteria is not only a cause of illness but a cure for it. Showing that not all bacteria is bad will show people that everyday goods are used by bacteria to form the goods we eat and use almost everyday.



c) The equiptment needed includes the 6 plastic tubes, pippets, plastic ring swabs, E.coli bacteria, milk, and a working BRAIN. Some of the key steps which include making sure the tubes dont get cross-contaminated. But, thats why you have to many controls to make sure you didnt accidently get some of the yogurt bacteria in the other tubes.



3. Procedure:
Have 6 tubes of milk that are labeled with varying to different controls....add ampicillin to tube "yogurt+amp"...using a new isolation loop dip into yogurt and swirl into "pos control"...using the same loop dip into "yogurt+amp"...ideentify two colonies on the agar plate and number them 1 & 2...using a fresh incoluation loop, pick the colony #1 and transfer it into the tube "yogurt colony #1" and do the same with #2...using another fresh loop pick an E.coli and put it in tube "E.coli"...place tubes in an incubator. Placing the tubes in the incubator help the bacteria duplicate and also helps the yogurt bacteria duplicate and form yogurt.

YOGURT LAB VIDEO:
http://www.youtube.com/watch?v=PbUoAO8hvcA

5. Discussion:

a) The data we recieved and got from the experiment was different then our hypothesis. Even though we all had a variety of hypothesis's we all came to the conclusion of the yogurt lab was going to be a success. In going through the lab we results came in; which were not as we expected. The yogurt in the tube that used yogurt bacteria wasnt as thick as we thought it would be and ended up smelling a little like yogurt (soury, kind of hard to describe). The control which were the other tubes turned out the way we guessed by the milk turning sour and smelling horrible (especially the E.coli). By using the control we provided evidence that not all bacteria form to make yogurt.

b) In the process of doing the lab, we had some errors that were made along the way which are in the video in the link above. Including in the vortex part where we didnt really hold onto the tubes of milk as they were swished around. Also the fact that we dropped the PH tester paper on the ground for one of the tubes and then having to wipe the yogurt bacteria on the sheet to get the excess off.