Forensic Files: The Don Strikes Again

Intro

DNA can be very useful for many things varying from Crime to Paternity.  DNA profiling, or fingerprinting can be used to analyze suspects, but it can also be used to solve an uncertain pregnancy.  A method called Restriction Fragment Length Polymorphism (RFLP) is currently preferred in the forensic world.  RFLP provides a banding pattern on Restriction Sites on the individuals DNA.  Restriction enzymes are a natural, bacterial defense mechanism.  A Restriction Enzyme is like a small pair of molecular scissors.  Restriction Enzymes destroy DNA from invading viruses or bacteriaphages.  Restriction enzyems are used to cut DNA strands in Agarose gel electrophoresis.  Agarose gel electrophoresis separates DNA fragments by size.  DNA is negatively charged, so it will be attracted to the positive anode at the other end of the gel.  Since everyone has different DNA, scientists have to create a way to locate, identify, and compare DNA of different individuals.  Scientists probe the DNA so that they can identify a particular strand of DNA.  This is called a "Radioactive Tag".  The tag can also be used to demonstrate genotype similarites between individuals. The size of the fragments reflect variations in individual's DNA.  Evidence for DNA fingerprinting can be collected from body tissues, bodily fluids(blood semen), hair follicles, etc.    

Procedure
PT 1.
1. Label 6 micro test tubes CS, S1-S5
2. Pipette 10 microliters of each DNA sample into the micro test tubes
3. Pipette 10 microliters of Enzyme Mix into the bottom of each tube
4. Microcentrifuge the tubes to both mix the two substances and make them all at the bottom of the micro test tube.
5. Place tubes into large volume of water heated to 37 degrees C.

PT 2.
1. Centrifuge micro test tubes to bring all liquid to the bottom
2. Add 5 microliters of loading dye to each micro test tube.
3. Remove Agarose Gel and plastic wrap
4. Place Agarose gel into electrophoresis apparatus.  Fill electrophoresis chamber with 1x TAE buffer to cover the gel, using approx. 275 mL of buffer for Bio-Rad mini-Sub Cell, horizontal electrophoresis chamber.
5. Using separate tip for each tube, load 10 microliters from the M tube, and 20 microliters from all of the other tubes. (CS, S1-S5).
6. Place lid back onto electrophoresis chamber, hook up the correct jacks with the correct colors (Red to Red, Black to Black).
7. Turn on power to 200V for 12 minutes.
8. Stain gel and record results.

DONE!

Results/Observations

Bioengineered: The Death of Petroleum

Intro

This lab involves using a Biofuel Enzyme Kit to create our own biofuels.  Through the use of certian enzymes, we will be able to create an alternate fuel source that is more efficient and better for the enviornment than burning petroleum products.  Ezymes are generally proteins and what they do is speed up the reaction rate.  Though enzymes are very effective, changes in salinity and pH of the substance can decrease the enzyme's effectiveness greatly.  In this lab, we are trying to extract the cellulose from plant cells and harvest that for energy.  In order to do this, we need to use the enzyme, cellobiase.

Procedure

Reaction Rates with/without Enzymes

1. Find your 15ml conical tube of Stop Solution and label it that

2. Find cuvettes and label 5 of them E1-E5

3. Put 500 microliters of stop solution into each cuvette

4. Label 2 empty conical tubes, Control and Enzyme Reaction

5. Pipet 2ml of 1.5 mM substrate into 15 ml conical tube labeled Enzyme Reaction

6. Pipet 1ml of 1.5mM Substrate into Control conical tube

7. Pipet 500 microliters of buffer into Control conical tube and gently mix.
8. After stirring remove 500 microliters of solution and add it to cuvette labeled start
9. Pipet 1mL of enzyme into 15mL conical tube, gently mix then START TIMER
10. At times indicated extract 500 microliters from solution and add it to correct cuvette containing the stop solution

E1=1min
E2=2min
E3=4min
E4=6min
E5=8min

11. After enzyme samples collected, remove 500 microliters of solution from Control and add it to cuvette labeled End
12. Analyze samples and clean supplies,  Hey you've just created a Biofuel!

Mushroom extracts Increase Reaction Rate??
1. Weigh out approx. 1 gram of mushroom
2. Add mushroom to mortar and pestel, and for every gram of mushroom, 2 mL of extraction buffer will need to be added.
3. Using pestel, grind mushroom to "slurry"
4. Add at least 250 microliters of Mushroom extract into a 1.5 mL microcentrifugal tube and place tube in centrifuge for 2  minutes
5. Label cuvettes 1-6.
6. Pipet 500 microliters of stop solution into each cuvette
7. Label 15mL conical tube with type of mushroom and then pipette 3 mL of substrate into the tube
8. Pipette 250 microliters of enzyme extract into 15 mL conical tube of Substrate.  START TIMER
9. At times remove 500 microliters of mushroon/enzyme into cuvette with stop solution.

1=1min
2=2min
3=4min
4=6min
5=8min

10. Analyze result, and clean materials that can be reused.  Your done!

Results/Discussion

From my observations, the solution in the cuvettes got more yellow as the time got longer.  This is due to more time the enzyme has to react with cellulose reaction.  Other than that, I thought that this lab was somewhat fun, but almost pointless because we didn't get to do anything with the finished product.  But anyway, it was interesting to see how enzymes can break through plant cells to collect that vital energy: glucose.



DNA: Extraction Point

Intro

DNA is a molecule located in all living things and its job is to contain special genetics that account for everything that makes you, you.  DNA is composed in a double helix roller coaster looking thing.    There are rungs between the two helix strands called bases.  There are 4 bases and they pair together as such, A-T G-C.    Cells called genes receive messages from the DNA to make proteins for certain characteristics in your body.  DNA travels throughout your body in mRNA molecules.  THese molecules then go to the ribosomes which actually create the proteins. 

The purpose of this particular experiment is to extract our own DNA from cheek cells, then after following various steps, the DNA will be revealed and then put into a necklace.  

Procedure

1. Loosen cheek cells with teeth

2. Swish saline solution in your mouth for 30 seconds

3. Spit into sterile tube

4. Insert the lysis buffer into saliva cell mixture to break the Cell membrane in order to extract DNA

5. Using micro-pippete, insert protease into tube in order to break down the proteins in the DNA (Histone) and for the DNAse.

6. Invert tube gently several times

7. Place tube in hot water bath for 5 minutes

8. Pour approximately 10 mls of Cold Ethanol into tube.

9. Cap tube and leave for 5 minutes

10. Slowly and gently invert tube and Observe DNA.  YEA!!! :)

Results/Observations

I thought this experiment was very cool.  When I tilted the tube after the cold ethanol, and the DNA appeared, I was ecstatic.  The DNA looked very similar to clear spaghetti.   Overall, a very easy and fun experiment.  

Koch's in the Kitchen: Yogurt postulates

Introduction

In the 1800s many scientists knew that bacteria existed, but they could not prove that the bacteria were the cause of the many appearing diseases and discomforts.  A scientist named Robert Koch developed 4 postulates that described how these new bacteria behaved.

1. The microorganism must be found in all organisms suffering from the disease, but not in healthy organisms.
2. The microorganism must be isolated from a diseased organism and grow in pure culture.
3. The cultured microorganism should cause disease when introduced into a healthy organism.
4. The microorganism must be again isolated from the inoculated, diseased experimental host and identified as identical to the original specific causative agent.

Many people think of disease when they hear the word Bacteria, but in fact, only a small percentage of the bacteria in the world are harmful.  This means that there are billions upon billions of different types of bacteria in the whole world.  Bacteria actually have their own domain, just like Eukarya and Archaea.  Bacteria are single celled microorganisms that actually play a giant role on our planet because they can do everything.  There are so many different types of bacteria that have different jobs just like people.  There are 3 main shapes of Bacteria, coccus (spherical), bacillus (rod-shaped), and spirillum (spiral).  Bacteria reproduce by binary fission, or copying itself and then splitting.   Bacteria also require food, just like any other living organism.  Bacteria are the ones responsible for eating sugars and then breaking them down into enzymes.


The purpose of this lab is to both demonstrate how not all bacteria is bad, case and point: yogurt.  And to reveal how little tiny single-celled microorganisms can create something so unique with very little material.  Because this technique for creating yogurt is so simple, yet so effective, large companies can utilize this and make yogurt for a cheap price and sell the person the container.  This may teach some people that it is much more cost effective to make their own yogurt with whatever fruits or flavors they desire.

When creating yogurt it is very important to label all important LAB materials, i.e. test tubes, petri dishes,    

etc.   Also it is important that all of the measurements are as accurate as possible.

Procedure

1. Fill 4 test tubes with milk.
2. Label each as follows, Negative Control (Milk), Positive Control (Milk), Positive Control + Ampicillin, E. Coli.
3. For the Negative Control, just leave as is.  For Positive Control, insert yogurt with plastic dowel and stir.  For Ampicillin, use a scientific dropper to insert the Ampicillin.  For E. Coli, scrape the cultures and drop it into the milk.
4.  Stir all of the tubes in a vortex stirrer.
5. Place all tubes into an incubating container of water at 37 degrees C.
6. Wait 24 hours and view results

Discussion 

After removing the tubes from the body temperature water, I observed each tube carefully to recognize which ones had created yogurt, and which had not.  From my results, the positive control did yield yogurt and so did the test tube with the Ampicillin.   The E. Coli and the Negative control though just stayed the same as they were before the incubator.  During the experiment I found that putting less yogurt on the plastic swab allowed me to insert it into the milk tube easier.