The Truth Concerning Queen ELISAbeth

Intro



Pregnancy Test Stick



In this lab, we will be testing and learning how to use the method of ELISA, or Enzyme Linked Immunosorbent Assay, to determine if we have been exposeded to a certain disease.  ELISA works by placing antigens into a container. Then primary antibodies are injected and they bind to the antigens. After the primary antibodies have binded, secondary antibodies with an enzyme are also injected, and the secondary antibodies bind to the primary.  Then a colorless substrate is added and it reacts with the enzyme. If the disease is present, then the color will change in the liquid.

Procedure

1. Label a yellow tube and a plastic transfer pipet with your initials.

2. Use the pipet to transfer all your "bodily fluid" sample into the tube of another student.  Gently mix the samples, then take back half of the shared sample (750 microliters) to your own tube.  Write down the name of the student next to "Sharing Partner #1".

3. When instructed to do so, repeat the sharing protocol two more times.  Discard this transfer pipet after this step.

4. Label your 12-well strip.  On each strip label the first 3 wells with a "+" for the positive controls and the next wells with a "-" for the negative controls.  Label the remaining wells with your and your lab partner's initials. (3 wells each). 

5. Use a fresh pipet tip to transfer 50 µl of the positive control (+) into the three “+” wells.

6. Use a fresh pipet tip to transfer 50 µl of the negative control (–) into the three “–” wells.

7. Transfer 50  µl of each of your team’s  samples from step 3 into the appropriately initialed three wells, using a fresh pipet tip for each sample.

8. Wait 5 minutes while all the proteins in the samples bind to the plastic wells.

9. WASH:

a. Tip the microplate strip upside down onto the paper towels, and gently tap the strip a few times upside down.  Make sure to avoid samples splashing back into wells.

b. Discard the top paper towel.

c. Use a fresh transfer pipet to fill each well with wash buffer, taking care not to spill over into wells. Note: the same transfer pipet is used for all washing steps.

d. Tip the microplate strip upside down onto the paper towels and tap.

e. Discard the top 2–3 paper towels.

10. Repeat wash step 9.

11. Use a fresh pipet tip to transfer 50 µl of primary antibody (PA) into all 12 wells of the microplate strip.

12. Wait 5 minutes for the antibodies to bind to their targets.

13. Wash the unbound primary antibody out of the wells by repeating all of wash step 9 two times.

14. Use a fresh pipet tip to transfer 50 µl of secondary antibody (SA) into all 12 wells of the microplate strip.

15. Wait 5 minutes for the antibodies to bind to their targets.

16. Wash the unbound secondary antibody out of the wells by repeating wash step 9 three times.

17. Use a fresh pipet tip to transfer 50 µl of enzyme substrate (SUB) into all 12 wells of the microplate strip.

18. Wait 5 minutes. Observe and record the results.

Phish Food For Thought

Intro

The connection between DNA, RNA, and Protein is not a foreign one, yet many people do not understand how organisms with completely different shapes and sizes, can be extremely similar in their DNA and Protein. In addition, a signaificant percent of people believe that the complexity of an organism relies on how large the total genome is of that organism.  They are simply wrong.  For example, a rat and a human are almost 99% similar in their DNA, yet they obviosly look nothing alike.  The realization by scietists that there is a large and diverse field of proteins, has started the study coined Proteomics.   Proteomics is often considered the next step after genomics, as they sound similar.  Proteomics mostly focuses on protein structure and function, and it is also, more complex than genomics.  An organization called Human Proteome Organization (HUPO), is an organization trying to catalog all human proteins and ascertain their functions and interactions.

In the end, we found that all of the food that we brought in (carrots) were in fact Genetically Modified.  This lab was definitely worthwhile because it really opened my eyes to how many foods are actually genetically manufactured.  Although the food may not taste any different, I now know that it is there.

PCR: A Tale of Two Genes

Intro

The blueprint that controls a large portion of our appearance, behavior, and tendencies is called our Genome.  The gonome is composed of DNA and is our hereditary code.  One method of expanding and cpying our genome is called PCR, or Polymerase Chain Reaction.  Essentially, PCR allows us to produce larege quatities of DNA in a test tube , starting from only a trace ammount.  Trace ammounts can be anything from a drop of blood, a hair follicle, or even a cheek cell.  PCR has left an imprint on four main areas of genomic research: gene mapping, cloning, DNA sequencing, and gene detection. To use DNA for PCR, you have to extract living cells from your body.  Once done, you place desired cells into a micro test tube conataining IntstaGene Matrix Beads.  These beads are responsible for killing DNAse in the cell so that once the membrane in broken, the DNA isn't dammaged. 

Procedure

DAY 1: Cheek Cell DNA Template Preparation 

1. Label on 1.5 mL micro test tube with your initials.  Label one screwcap tube containing 200 microliters of InstaGene matrix with your initials.

(Rinse Out Mouth if Necessary)

2. Obtain a cup containing saline solution from your instructor.  Chew the insides of your cheeks gently, and pour the saline into your mouth and rinse vigorously for 30 seconds.  Expel the saline back into the cup.

3. Transfer 1 mL of your saline rinse into the micro test tube (NOT the screwcap tube) with your initials.  

(Save cup for step 5)

4. Spin your tube in a balanced centrifuge at full speed for two minutes.  At the bottom of the tube you should see a match-head sized pellet of whitish cells at the bottom of the tube.

5. After pelleting cells, pour off the saline into your cup being careful not to lose your pellet.

6. Re-suspend pellet by flicking or vortexing the tube so no clumps remain.  

7. Using a 2-20 micro-pipette, set it at 20 microliters and transfer all of your suspended cells into the screwcap labeled InstaGene.  Discard the flip-top tube.

8. Screw the cap tightly on the tube and shake or vortex the tube to mix the contents.

9. When all members have collected their cells, place tubes in foam micro test-tube holder, and incubate at 56 degrees C for 10 minutes in a water bath.  

10. Remove tubes, shake or vortex, and place the tubes in a boiling water bath (100 degrees C) for 5 minutes.

11. Remove tubes from boiling water bath and shake or vortex to re-suspend.  Pellet the matrix by spinning at 6,000 x g for 5 minutes.

12. Store screwcap in the refrigerator until the next day.

DAY 2: PCR Amplification (Use PCR tips today)

1. Obtain tube with your DNA template from the refrigerator.  Spin the screwcap tube for 2 minutes at 6,000 x g in a centrifuge. 

2. Label PCR tubes with your initials and place PCR tube in your PCR rack.

3. Transfer 20 microliters of the DNA template from the screwcap into the bottom of the PCR tube.  Be careful not to transfer any matrix beads into the PCR tube.

4. Put 20 microliters of Master Mix directly into the DNA template.  Cap tube tightly.  Mixture should be   yellow.

5. Place PCR tube into the thermal cycler.  

DAY 3: Electrophoresis of Amplified PCR Samples and Staining of Agarose Gels

1. Obtain PCR tube from the thermal cycler and place in the capless micro test tube.  Pulse-spin the tube for approx. 3 seconds.

2. Add 10microliters of PV92 XC loading dye into your PCR tube and mix gently.

3. Place an agarose gel in the electrophoresis apparatus.  Check that the wells of the agarose gels are near the black (-) electrode and the base of the gel is near the red (+) electrode.

4. Fill electrophoresis chamber and cover gel with 1x TAE buffer.  This will require approx. 275 mL of 1x buffer.

5. Using a clean tip for each sample, load the samples into 8 wells in the following order:

Lane	Sample	Load Volume
1	MMR (DNA standard)	10 microliters
2	Homozygous (+/+) control	10 microliters
3	Homozygous (-/-) control	10 microliters
4	Heterozygous (+/-) control	10 microliters
5	Student 1	20 microliters
6	Student 2	20 microliters
7	Student 3	20 microliters
8	Student 4	20 microliters

6. Secure the lid on the gel box.  Red-Red, Black -Black.  Connect electrical leads to power supply.

7. Turn on power and electrophorese your samples at 200V for 3 min.

Staining of Agarose Gel

When electrophoresis is complete, remove lid and place gel into tray.

DONE!! 

Genetically Modified: A Plant's Life

Intro

Today, most foods that are sold in grocery stores are Genetically Modified.  Genetically Modified Organisms, or GMO's, differ from normal or organic organisms in that they have been specially designed to grow or be a certain way.  GMO's are achieved by scientists placing a gene of intrest into the DNA of a particular organism.  Farmers have been genetically modifying crops for centuries to encourage specific traits, such as high yield.  Genes that are inserted into a plants DNA do not have to even be plant genes at all.  Although GM crops seem like a positive alternative, many people would disagree.  They say that a super weed or a super insect might be the unintended result, and that weed or insect may be resistant to the pesticides used today. Also, genetically modifying crops may introduce alergens into that food that the buyer might not be aware of.  The Technique that we will be using for this Lab involves Polymerase Chain Reaction, or PCR.  PCR essentially is a copier for DNA.  For instance, a scientist can have trace amounts of a certain fragment of DNA, and after using PCR, they will have millions of copies of that desired strand of DNA.  PCR can be used in a variety of situations, from medical diagnosis, to crime scene investigations, and even for aiding sequencing the human genome.

Procedure

DAY 1:

1. Find your screwcap tubes and label one "Non GMO", and the other "test".

2. Weigh out .5-2g of certified non-GMO food and put it in the mortar and pestle.

3. Add 5 mL of distilled water for every gram of food.  The ratio of food to water is achieved from this equation: Mass of Food=____g x 5 = ____ mL

4. Grind with pestle for at least 2 min to form a slurry.

5. Add 5 volumes of water again and mix or grind further with pestle until smooth enough to pipet.

6. Pipet 50 microliters of ground slurry to the screwcap tube containing 500 microliters of InstaGene labeled "non-GMO".

7. Repeat steps 2-5 to prepare the test food sample.

8. Pipet 50 microliters of ground test food slurry to the screwcap tube labeled "test".

9. Shake or flick the non-GMO food and test food InstaGene tubes and place tubes in 95 degree Celsius water bath for 5 minutes.

10. Place tubes in a centrifuge in a balanced conformation and centrifuge for 5 minutes at max speed.

11. Store tubes in a refrigerator until next lesson.

DAY 2:
1. Number PCR tubes 1-6 and initial them.  The numbers should correspond to the following tube contents:

Tube Number	Master Mix	DNA
1 (Carrot)	20 Microliters Plant MM (green)	20 Microliters Non-GMO food control DNA
2 (Carrot)	20 Microliters GMO MM (red)	20 Microliters food control DNA
3 (Corn Flour)	20 Microliters Plant MM (green)	20 Microliters Test Food DNA
4 (Corn Flour)	20 Microliters GMO MM (red)	20 Microliters Test Food DNA
5 (Control)	20 Microliters Plant MM (green)	20 Microliters GMO Positive Control DNA
6 (Control)	20 Microliters GMO MM (red)	20 Microliters GMO positive control DNA

2. Place each tube in a capless microtube adaptor and place in the foam float on ice.
3. Refering to the table and using a fresh tip for each addition, add 20 microliters of the indicated Master Mix to each PCR tube.
4. Refering to the table and using a fresh tip for each tube, add 20 Microliters of the indicated DNA to each PCR tube, be sure to avoid the InstaGene pellet at the bottom of the tubes.  Mix by pipetting gently up and down.
5. When instructed, place PCR tubes in thermal cycler.

DAY 3:
1. Set up your gel electrophoresis apparatus as instructed.
2. Obtain your PCR tube from the thermal cycler and place in the capless microtube adaptor.  Pulse spin for -3 seconds.
3. Using a fresh tip each time add 10 microliters of Orange G loading dye to each sample and mix well.
4.  Load 20 Microliters of the molecular weight ruler and 20 microliters into your gel in the order indicated below:

Lane	Sample	Load Volume
1	Sample 1: Non-GMO food control with plant primers	20 Microliters
2	Sample 2: Non-GMO food control with GMO primers	20 Microliters
3	Sample 3: Test Food with Plant Primers	20 Microliters
4	Sample 4: Test food with GMO primers	20 Microliters
5	Sample 5: GMO positive DNA with plant primers	20 Microliters
6	Sample 6: GMO positive DNA with GMO primers	20 Microliters
7	PCR Molecular Weight Ruler	20 Microliters
8	Leave Empty	-

5. The run time and voltage will depend on the type of gel you are running.  Run an agarose gel for 30 mins at 100 V and run a polyachrylamide gel at 200 V for 20 minutes.
6. Stain in Fast Blast DNA stain.  Refer to specific instructions depending on gel type.

Results

Upon reviewing my own results, it turns out that my test food was a genetically modified food.  The corn Flour also, was genetically modified.   All in all, this lab was very beneficial and it opened my eyes up to the fact that almost all of the food on the market nowadays is genetically modified. 

The Jellyfish Chronicles: A Glowing Transformation

Intro

This lab invovles a process known as genetic transformation.  Genetic transformation means a change caused by genes, and involves the insertion of a gene into an organism in order to change the organisms trait.  In this lab we ware transforming the bacteria that codes for Green Fluorescent Protein (GFP).  In nature, this protein can be found in the bioluminescent sea-jelly Aequorea victoria.   Genetic transformation includes inserting new DNA into E.Coli cells.  Bacteria often contain one or more small circular pieces of DNA called plasmids.  Plasmid DNA usually contains genes for more than trait.  Scientists use a process called genetic engineering to insert coding for new traits into a plasmid.  The new genetically engineered plasmid can be used in genetically transforming bacteria to give them the new trait.

Procedure

1. Label 1 micro test tube +pGLO and another -PGLO.

2. Using a sterile transfer pipette, transfer 250 microliters of transformation solution (CACl2)

3. Place tubes on ice for 5 minutes.

4. Using a sterile loop, pick up one colony of bacteria from the starter plate.  Place bacteria into +pGLO tube.  Make sure all of the bacteria gets into tube.place tube in rack in ice.  Do the same for -pGLO.  

5. Add 10 microliters of plasmid DNA directly to liquid in +pGLO tube only.  Flick tube to mix.  Close tube and return to rack on ice.  Do not add plasmid DNA to -pGLO tube.  

6. Incubate tubes on ice for 10 minutes.

7. While tubes are on ice, label four Agar plates: LB/Amp plate +pGlO, LB/Amp/Ara +pGLO, LB/Amp -pGLO, LB -pGLO. 
8. Heat Shock.  Using foam rack, transfer both (+) (-) pGLO's into water bath set at 42 degrees C for exactly 50 seconds.  After 50 seconds, place tubes back on ice.  For best results, the change from heat to cold must be quick.  Incubate tubes on ice for 2 minutes.
9. Remove tubes from ice and with a new sterile pipette add 250 microliters of LB nutrient broth in both +pGLO and -pGLO tubes.
10.  Using sterile pipette, pipette 100 microliters of the transformation and control suspensions onto the appropriate plates.
11. Using a new sterile loop for each plate, spread the suspensions evenly around the surface of Agar.
12. Stack plates upside down and place them into the 37 degree C incubator until the next day.

Results

Control 

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.