The Diseased at Acalanes

For this lab we will be using the technique of PCR to extract the DNA to make it able to get he certain parts of the DNA we are searching for. In 1983, Karry Mullis at Cetus Corporation developed the molecular biology technique for this type of research which is called PCR. The reason we use PCR is that it produces a abundance of DNA in a test tube starting from a little bit of it. So basically it is a way of amplifying the DNA sequence, gene of interest. With this we can use the genomic DNA and just from a DNA sample no matter how small it can be made bigger amount so it has enough DNA to study with.

PCR is the best for genetic research, gene mapping, cloning, DNA sequencing, and gene detection and will be used in medical diagnostic. It will be a tool to detect the mutations that cause the genetic disease, also in criminal investigations and court of law to help identify the suspects, sequencing the human genome.



Procedure:

Day 1
Extract the cheek cells by chewing the in inside of your cheeks for 30 seconds
get the DNA and put the InstaGene Matrix inside of the small tube
put in the heat bath for 10 mins
heat then in the 94 degrees Celsius water bath
vortex the tube for 1 min
extract the water on the top of the tube and leave the clump on the bottom

Day 2
Add the loading dye to the DNA sample
and also add the controls of +/+ -/- +/- and marker to the gel in the last lane
add the DNA samples from the students samples
Run the gel *running to red for 15 mins
transfer gel when done to a dye stain tray
go to the back and add the blue stain to stain the DNA in the gel
put on rocker to marinade the gel over the weekend

Day 3 


Results:


i figured out what we were looking for was the having the so called "disease" and i ended up having the disease gene and only one person in our group was able to not have that gene.


Discussion:
a) i had no idea of what to hypothesis but we tested for the presence of an enzyme and the odds were high to have it show up as positive so i guessed i would be so called "diseased"

b) DONT chew too hard or else you will start to bleed from your cheeks and also make sure you do it long enough so you have enough cells.

GM or not to GM that is the question....

As the farmable land is disappearing agriculitural specialists are looking for an alternative to produce enough food to feed the growing populations. Also concerned about the use of pestocides and herocides on the population and the longterm effects could do to human health. The GM "genetically modified" plants can solve these problems but have been under great controversy of what it can and will do in the future of human health as well.

Genetic plants arent new but farmers have gradually started to used the GM plants for these specific traits. These preffered genes are being put into the plants and also having the pesticides put into the plants so the use of sprays are cut by a huge amount. This resistance gene is being taken from a soil bacterium called Bacillus thuringiensis being put into the plant's genome.

People are not liking the GM plants because of the potentcial to create super-weeds through the cross polination with herbocide-resisant crops or also super-bugs will evolve over time to resist the pesticides. But the trade off is that the arguement of possibly better for the environment because of less chemicals being put into the atmosphere.

The GMO debate need to be proven by testing the foods found in grocery stores for the presence of GMO-derived products. This can be done by using an antibody-based test another is polymerase chain reaction (PCR) to look at the sequence common in GM foods. DNA is more resistant  than proteins in processing and can be extracted form even highly processed foods.

Playing god? Thats childs play, being god is a different story.....

Intro:
This lab involves us to perform "genetic tranformation" which invloves a pice of DNA which provides the instructions and it really means for us to change the genetic information. This entales us to take a gene and insert it into another organism inorder to change the organism's traits. The use of genetic transformation is is used in biomediation which is consisting of oilspill soaking bacteria and also it's used in agriculture. The use in agriculture is abundant and is used to prevent disease killing and to keep certain traits in plants to make a perfect crop. Its is in medicine as well, the diseases caused by defective genes are beginning to be treated by gene therapy by transformaing the sick persons cells with healthy coppies of the defactive gene to help with the disease.

The experiment we will be conducting involves us to transform bacteria with the gene with (GFP) or Green Fluorescent Protien which is in the sea jelly Aequorea Victoria. The bacteria will have this gene and express it under ultraviolet light and cause them to glow a brilliant green.

Using the plasmid we will learn how to be able to tranfer thes genes from one organism to the next and have a funn time doing it. The bacteria contain circular pieces of DNA called plasmids these contain genes for one or more of the organisms traits that could be important to the bacteria's survival.

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.