Bio 05LA Fall Quarter 2013

Lab 4A

Lab 4A: Genetic Testing Laboratory Detection of Alu Sequences by PCR *

It is estimated that there are 30,00050,000 individual genes in the human genome. The true power of PCR is the ability to target and make millions of copies of (or amplify) a specific piece of DNA (or gene) out of a complete genome. In this activity, you will amplify a region within your chromosome 16. Amplifying the Target Sequence The recipe for a PCR amplification of DNA contains a simple mixture of ingredients. To replicate a piece of DNA, the reaction mixture requires the following components: 1. DNA template containing the intact sequence of DNA to be amplified which in this case is genomic DNA that will be extracted from your cheek cells. 2. Individual deoxynucleotides (A, T, G, and C) raw material of DNA 3. DNA polymerase an enzyme that assembles the nucleotides into a new DNA chain 4. Magnesium ions a cofactor (catalyst) required by DNA polymerase to create the DNA chain 5. Oligonucleotide primers pieces of DNA complementary to the template that tell DNA polymerase exactly where to start making copies 6. Salt buffer provides the optimum ionic environment and pH for the PCR reaction The two DNA primers provided in this kit are designed to flank a DNA sequence within your genome and thus provide the exact start signal for the DNA polymerase to zero in on and begin synthesizing (replicating) copies of that target DNA. Taq DNA polymerase extends the annealed primers by reading the template strand and synthesizing the complementary sequence. In this way, Taq polymerase replicates the two template DNA strands. PCR amplification includes three main steps, a denaturation step, an annealing step, and an extension step (summarized in Figure 1). In denaturation, the reaction mixture is heated to 94C for 1 minute, which results in the melting or separation of the double-stranded DNA template into two single stranded molecules. The DNA templates must be separated before the polymerase can generate a new copy. The high temperature required to melt the DNA strands normally would destroy the activity of most enzymes, but Taq polymerase is stable and active at high temperature. During the annealing step, the oligonucleotide primers anneal to or find their complementary sequences on the two single-stranded template strands of DNA. In these annealed positions, they can act as primers for Taq DNA polymerase. Binding of the primers to their template sequences is also highly dependent on temperature. In this lab exercise, a 60C annealing temperature is optimum for primer binding. During the extension step, the job of Taq DNA polymerase is to add nucleotides (A, T, G, and C) one at a time to the primer to create a complimentary copy of the DNA template. During polymerization the reaction temperature is 72C, the temperature that produces optimal Taq polymerase activity. The three steps of denaturation, annealing, and extension form one cycle of PCR. A complete PCR amplification undergoes 40 cycles. The entire 40 cycle reaction is carried out in a test tube that has been placed into a thermal cycler. The thermal cycler contains an aluminum block that holds the samples and can be rapidly 1

Bio 05LA Fall Quarter 2013

Lab 4A

heated and cooled across broad temperature differences. The rapid heating and cooling of this thermal block is known as temperature cycling or thermal cycling.

5' 3'

3' 5'

Denature strands at 94C

5'

3' 5'

3'

Anneal primers at 60C

(Taq polymerase recognizes 3' ends of primers)

5'

3' 5' 3'

Primer

Taq polymerase

3'

5'

Primer
5'

3'

Extend at 72C
(Synthesize new strand)

5' 3' 5'

3'

5' 3'

3' 5'

Repeat cycle 40 times

Fig. 1. A complete cycle of PCR.

The Target Sequence The human genome contains small, repetitive DNA elements or sequences that have become randomly inserted into it over millions of years. One such repetitive element is called the Alu sequence. This is a DNA sequence about 300 base pairs long that is repeated almost 500,000 times throughout the human genome. The origin and function of these repeated sequences is not yet known. In this laboratory activity you will look at an Alu element in the PV92 region of chromosome 16. This particular Alu element is dimorphic, meaning that the element is present in some individuals and not others. Some people have the insert in one copy of chromosome 16 (one allele), others may have the insert in both copies of chromosome 16 (two alleles), while some may not have the insert on either copy of the chromosome (Figure 2). The primers in this kit are designed to bracket a sequence within the PV92 region that is 641 base pairs long if the intron does not contain the Alu insertion or 941 base pairs long if Alu is present. This increase in size is due to the 300 base pair sequence contributed by the Alu insert.

Bio 05LA Fall Quarter 2013

Lab 4A

When your PCR products are electrophoresed on an agarose gel, three distinct outcomes are possible. If both chromosomes contain Alu inserts, each amplified PCR product will be 941 base pairs long. On a gel they will migrate at the same speed so there will be one band that corresponds to 941 base pairs. If neither chromosome contains the insert, each amplified PCR product will be 641 base pairs and they will migrate as one band that corresponds to 641 base pairs. If there is an Alu insert on one chromosome but not the other, there will be one PCR product of 641 base pairs and one of 941 base pairs. The gel will reveal two bands for such a sample.

Fig. 2. The presence or absence of the Alu insert within the PV92 region of chromosome 16.

PV92
ALU

Genotype

DNA Size of PCR Products

ALU

Homozygous (+/+)

941 base pairs

Homozygous (/)

641 base pairs

ALU

Heterozygous (+/)

941 and 641 base pairs

(bp) 1,000 700 500 200 100

Fig. 3. Electrophoretic separation of DNA bands based on size. EZ Load DNA molecular mass ruler, which contains 1,000 bp, 700 bp, 500 bp, 200 bp, and 100 bp fragments (lane 1); two homozygous (+/+) individuals with 941 bp fragments (lanes 2, 6); three homozygous (/) individuals with 641 bp fragments (lanes 3, 5, and 8), and two heterozygous (+/) individuals with 941 and 641 bp fragments (lanes 4 and 7).

Bio 05LA Winter Quarter 2013

Lab 4A

Electrophoresis separates DNA fragments according to their relative sizes. DNA fragments are loaded into an agarose gel slab, which is placed into a chamber filled with a conductive buffer solution. A direct current is passed between wire electrodes at each end of the chamber. DNA fragments are negatively charged, and when placed in an electric field will be drawn toward the positive pole and repelled by the negative pole. The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily than larger ones. Over a period of time, smaller fragments will travel farther than larger ones. Fragments of the same size stay together and migrate in what appears as a single band of DNA in the gel. In the sample gel above (Figure 3), PCR-amplified bands of 941 bp and 641 bp are separated based on their sizes. Lab Period 1 Isolation of Genomic DNA and PCR To obtain DNA for use in the polymerase chain reaction (PCR) you will extract the DNA from your own living cells. It is interesting to note that DNA can be also extracted from mummies and fossilized dinosaur bones. In this lab activity, you will isolate DNA from epithelial cells that line the inside of your cheek. To do this, you will rinse your mouth with a saline (salt) solution, and collect the cells using a centrifuge. You will then boil the cells to rupture them and release the DNA they contain. To obtain pure DNA for PCR, you will use the following procedure: The cheek cells are transferred to a microcentrifuge tube containing InstaGene matrix. This particulate matrix is made up of negatively charged, microscopic beads that chelate, or grab, metal ions out of solution. It traps metal ions, such as Mg2+which are required as catalysts or cofactors in enzymatic reactions. Your cheek cells will then be lysed, or ruptured, by heating to release all of their cellular constituents, including enzymes that were once contained in the cheek-cell lysosomes. Lysosomes are sacs in the cytoplasm that contain powerful enzymes, such as DNases, which are used by cells to digest the DNA of invading viruses. When you rupture the cells, these DNases can digest the released DNA. However, when the cells are lysed in the presence of the chelating beads, the cofactors are adsorbed and are not available to the enzymes. This virtually blocks enzymatic degradation of the extracted DNA so you can use it as the template in your PCR reaction. You will first suspend your isolated cheek cells in the InstaGene matrix and incubate them at 56C for 10 minutes. This preincubation step helps to soften plasma membranes and release clumps of cells from each other. The heat also inactivates enzymes, such as DNases, which can degrade the DNA template. After this 10 minute incubation period, place the cells in a boiling (100C) water bath for 5 minutes. Boiling ruptures the cells and releases DNA from their nuclei. You will use the extracted genomic DNA as the target template for PCR amplification.

Chromosome 16: PV92 PCR Informatics Kit Manual which is a product of Bio-Rad. Duplication of any part of this document is permitted for classroom use only

*The introductory information and protocol has been adapted from Biotechnology Explorer-

Bio 05LA Winter Quarter 2013

Lab 4A

Procedure
Lab Period 1 - Cheek Cell DNA Template Preparation
1. Obtain a cup containing saline solution from your instructor and go to your lab bench. Pour the saline into your mouth and rinse vigorously for 30 seconds. Expel the saline back into the cup.

2. Label the 1.5 ml micro test tube (a) on the bench with your seat number.

3. Transfer 1 ml of your saline rinse into the micro test tube (a) using a 1 ml pipet.

(a)

(b)

4.

Along with the rest of your classmates, spin your tube in a balanced centrifuge at full speed for 2 minutes. When the centrifuge has completely stopped, remove your tube. You should see a match-head sized pellet of whitish cells at the bottom of the tube. If you dont see a pellet of this size, decant the saline, refill your tube with more of your oral rinse, and repeat the spin.

(a)

Centrifuge

5. After pelleting your cells, pour off the saline. Being careful not to lose your pellet, blot your tube briefly on a paper towel or tissue. Its OK for a small amount of saline (< 50 l, about the same size as your pellet) to remain in the bottom of the tube.

6. Resuspend the pellet by vortexing or flicking the tube so that no clumps of cells remain. 7. Obtain a screw cap tube containing 200 l of InstaGene matrix from your TA. The tube number should match your seat number. Using a 1ml pipet, transfer all of your resuspended cells to the screwcap tube.

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

Bio 05LA Winter Quarter 2013

Lab 4A

9. When all members of your team have collected their samples, place the tubes in the micro test-tube holder and incubate at 56C for 10 minutes in a water bath. At the halfway point (5 minutes), shake or vortex the tubes gently, then place back in the 56C water bath for the remaining 5 minutes.

Water bath 56C, 10 min Very Hot!

10. Remove the tubes, shake or vortex, and place the tubes in 100C dry bath. Incubate at 100C for 5 minutes. Be careful, the block is very hot!

Dry bath 100C, 5 min

11. Remove the tubes from the 100C dry bath and shake or vortex the contents to resuspend. Pellet the matrix by spinning at full speed for 5 minutes in a centrifuge.

Centrifuge
12. Place your screwcap tube in the TAs ice bucket. The TA will prepare your samples for PCR by transferring 20 l of your DNA sample to a PCR tube. The TA will then add 20 l of the master mix which contains the nucleotides, primers, magnesium ions, buffer and Taq Polymerase. The tubes are then placed in the thermal cycler for 40 cycles of amplification. Once the program has run, the PCR reactions will be frozen and stored until the next lab meeting.

Lab Period 2 Separation and Visualization of PCR Products

The PCR products you generated week 4 will be separated by gel electrophoresis. After thawing the PCR reactions your TA will add 10 l of loading dye. The TA will demonstrate the technique for loading samples into the wells of an agarose gel (A). The lid is then placed on the apparatus and the power cords are plugged into the power supply (B). Separation of the fragments will occur as the current flows through the gel. Note that the cords are color coordinated. The red cord represents the positive end of the field and the DNA will migrate toward that end of the gel (the dye front will be your visual indicator). After about 40 minutes at 120V the electrophoresis will be stopped and the gel transferred to a tray containing a concentrated staining solution (C). The DNA in the gel will bind the dye allowing visualization of the bands.

Can you determine whether you are homozygous (+/+), homozygous (-/-) or heterozygous? Genotype determined ____________________________________

Biology 05LA Fall Quarter 2013

Lab 4B page 1

LAB 4 -- HYPOTHESIS-BASED SCIENCE; AN INTRODUCTION TO THE HYPOTHETICO-DEDUCTIVE APPROACH TO PROBLEM SOLVING.

Science has been described as a way of knowing, that is, it is an activity whereby we learn about our world. This learning can be achieved by two different approaches. The first applies to inquiries in new or poorly understood areas and is referred to as discovery or descriptive science. Here, data is collected in the form of careful observations of a phenomenon of interest. This data is then carefully analyzed and recorded in a retrievable manner. As more and more data is collected, generalizations about this phenomenon can be made and our understanding thus increases. The type of reasoning by which this understanding was achieved is referred to as inductive because larger generalizations are derived from many smaller specific observations. In contrast, hypothesis-based science deals with questions in areas that are better understood. Here, problem solving begins with a well-defined question. The next step involves consideration of what is known about related phenomena and how we came to know these things. This information is then used as the basis for formulating a hypothesis, or possible answer to the question. The order in which this factual information is assembled is determined with the use of deductive reasoning. That is, it is ordered in a manner that places more general knowledge first and continues with information that is progressively more specific to the hypothesis being developed. There are two important concerns regarding hypothesis formation. First, hypotheses need to be falsifiable with an experimental test. Second, a workable experiment must be designed to make the test. Once a suitable hypothesis has been proposed and an experiment designed, a prediction is made about the expected outcome of the experiment given that the hypothesis is correct. This predictability is made possible by the deductive reasoning that was used in the formulation of the hypothesis in concert with an awareness of the experiment that will be performed. What is desirable here is an if/then relationship between the hypothesis and the prediction. This is presented as follows: if the proposed hypothesis is acceptable and the experiment is performed, then the predicted result should be attained. The experiment is then performed. If the prediction matched the experimental result, then the hypothesis is conditionally supported in light of the knowledge on which it was based. If the prediction did not match the experimental result, then there was a problem with the hypothesis, the experimental approach, or both. In either case, the hypothesis and/or the experiment should be reconsidered. In the real world of scientific research, it is not at all unusual for predictions not to match results in early efforts to answer a question. While this may seem discouraging to the new science student, the positive aspect of this approach is that it represents a learning process. That is, even with the failure of one iteration of the process, useful information has been gained for better focusing the questions, hypotheses, experiments, and predictions for the next round which should be more successful. In earlier editions of your text and this lab manual, the process described above is referred to as the Hypothetico-Deductive (H/D) approach to problem solving the origin of this terminology should be obvious from the above discussion. In Biology 5, we wish to demonstrate the practical value of the H/D approach by expecting the student to apply it to the investigative laboratories presented in this course. Some modifications of the procedure need to be made in order to make it applicable to work in the teaching lab. These are the subject of the following discussion. The H/D approach in Biology 5 laboratories. The major differences between the form of the H/D approach used by professional researchers and that which you will employ as beginning biology students are that the questions addressed by the lab exercises have already been defined as have the experimental protocols used for their study. These differences make the students job easier, but not trivial! Here is the amended procedure:

Biology 05LA Fall Quarter 2013

Lab 4B page 2

1) A fundamental demand for successfully applying the H/D approach is that the student must have attended all of the lectures associated with the lab exercise, read all of the notes taken from the lectures, and read all of the relevant assignments in the text. This requirement is directly comparable to the professional researchers need to have read all of the literature in scientific journals that pertains to their questions. 2) Next, it is time to carefully read (and reread as necessary) the exercise in the lab manual. You then must decide which experiments are associated with the different questions addressed by the lab exercise (these are found either at the end of this exercise or within the relevant lab exercise). 3) Now starts the task of formulating hypotheses (or proposed answers) for the questions. This process begins with the collection factual information relevant to the question. The sources for this information include your lecture notes, the text, and the lab write-ups in this lab manual. The challenge here is to organize this information deductively, that is moving in a progression from general to specific. This step is challenging; you should see your TA for help if you are having problems. Keep in mind that: a) hypotheses are possible answers/explanations. b) hypotheses reflect present knowledge about the subject area of the question. c) hypotheses should be expressed in terms that are testable by the experiments being done. That is, you should be able to make predictions of the outcome of your experiments given that your hypotheses are supportable. d) hypotheses can be eliminated but not confirmed with absolute certainty. 4) Next, you must come to understand the experimental strategy of the experiments you will be performing. In other words, you need to make sure that you understand the relationship between the data you will be collecting and the questions you are attempting to answer. In some cases, the relationship between your data and your hypotheses will be direct and straight forward. In others, some explanations are required to make this connection. This explanation is what we are calling experimental strategy. 5) At this point, the Bio 5 amendments end and the H/D process will continue as described above. Once again, predictions of the experimental results are made assuming that your hypotheses are supportable. Predictions are best presented in an if/then form; if my hypothesis is true and the experiment is done, then I should get this result. 6) Finally the experiment is performed and the result is compared with the prediction. If the result matches the prediction, then your hypothesis is supported. If the result differs from the prediction, you may need to reconsider your hypothesis, reevaluate your experimental technique, or both. A practical application of the H/D approach. The following is intended to help you see why this approach is so effective for problem solving and to give you a feeling for how we expect you to use this process for the presentation of your lab write-ups. This example from everyday life is derived from an experience which many of us have had. You get into your car, turn the key and nothing happens. Here, the question is obvious; why wont my car start? Before a possible answer (i.e. hypothesis) can be proposed, it is necessary to collect some information about how cars start because hypotheses need to be based upon available facts. To this end, you speak to your neighbor who teaches auto shop in the local high school. She tells you that the starter system works as follows: The key operates a switch that, when turned, directs electricity from the battery to the starter via a system of wires. This power does two things. First, it activates a solenoid which connects the starter motor to the engine. Second, it drives the starter motor which

Biology 05LA Fall Quarter 2013

Lab 4B page 3

actually turns the engine over and starts the engine. At this point you have plenty of information for proposing a few of the several possible hypotheses -- we will consider three of these. QUESTION: Why wont my car start?
Hypothesis
1. The battery is dead (i.e. it has no electrical charge).

Supporting fact(s)
Electricity from the battery is required to power the starter.

The experiment & Experimental strategy

The experiment: Attempt to turn on the lights. The experimental strategy: Since the battery powers most of the electrical systems in a car, a dead battery should also affect these systems (including the headlights).

Prediction
If the battery is dead, then the headlights wont work.

You now perform an experiment by attempting to turn on the lights. They come on. This result falsifies your hypothesis and you need to continue. However, this experiment was not a wasted effort. Now you know that electricity is available to run the starter.
Hypothesis
2. Electricity is not getting to the starter assembly.

Supporting fact(s)
Electricity is required for the starter to operate.

The experiment & Experimental strategy

The experiment: Connect a voltage meter to the power supply terminals on the starter. The experimental strategy. Since the experiment involves the direct measurement of electricity at the starter, the data that will be collected relates directly to the question.

Prediction
If electricity is not getting to the starter assembly, then the meter will not sense power at the starter when the key is turned.

The meter is connected to the starter and it shows that electricity is reaching it. This result falsifies your hypothesis and you need to continue. Once again, this experiment was not a wasted effort. Now you know that problem is probably with the starter assembly.
Hypothesis
3. The starter solenoid is not functioning.

Supporting fact(s)
The starter motor must be engaged with the engine for it to be able to start the engine. The solenoid engages the starter motor.

The experiment & Experimental strategy

The experiment: Replace the starter solenoid. The experimental strategy. Here the experimental data will relate directly to the question.

Prediction
If the starter solenoid is not functioning, then installation of a new one should allow the car to be started.

A new solenoid is installed and the car starts like new. Thus, your car would not start because the starter solenoid was defective. From this example you should see: The absolute necessity of learning as much as you can about the problem you are faced with before you attempt to formulate a hypothesis. The importance of presenting a hypotheses, supporting facts, experimental strategies, and predictions in the most concise manner possible -- This gives your problem solving effort the focus necessary to make it effective.

Biology 05LA Fall Quarter 2013

Lab 4B page 4

That an elaborate discussion of the experimental strategy is not required for experiments that provide data relating directly to the hypothesis. That the H/D approach is useful in the everyday world as well as in the laboratory.

Practical use of the H/D approach for the Write-Ups for investigative labs. Starting with the exercise on enzymes and continuing with the next lab on metabolism, you will be performing two truly experimental, investigative labs. As such they provide excellent opportunities for learning and appreciating the value of H/D approach as a process whereby we can expand our knowledge of biology. We consider this experience as one of the most important aspects of the curriculum of this course and your development as a science scholar! The guide that follows was prepared to help you with your weekly lab write-ups. The Rules: 1) While it is acceptable to discuss the lab write-ups with others, each student is expected to independently write his or her intros for submission and grading. 2) You must prepare the Introduction in advance of your weekly lab session. This is an essential step toward coming to lab adequately prepared.* A copy of your introduction (preparation guidelines follow) will be handed in to your TA at the beginning of the lab session on the day you are to perform the lab exercise. Late introductions will receive no credit. Also, you are reminded that you must perform the lab exercise to receive credit for its introduction. 3) Make sure that you understand what is expected of you before you begin. A major part of this exercise is to develop your ability to express yourself in a concise and focused manner. This takes time. 4) You are encouraged to have your TA preview and comment upon your introductions before they are handed in this is best done during your TAs office hours and not via email. 5) The introductions must be typed. 6) The results and discussion sections for these labs will be completed in the lab notebook. The processed data for the Enzyme (graphs and calculations) and Fermentation/Respiration (calculations) labs will be completed and handed in for grading at the times specified in the lab schedule.
* Adequate preparation also includes advanced preparation of any necessary data sheets for recording the data to be collected.

Preparation guidelines for the lab Introductions: The guide that follows provides specific guidelines for the preparation of your Introductions (and the Results and Discussion sections). The Introductions will be organized in the manner specified here or they will not be graded. The Introduction. For each of the questions listed for a particular lab exercise, you are expected to prepare separate introductory statements. Each of these should be organized as follows: Question #___: Here you should present the question you are addressing. 1) Your hypothesis: Insert here your hypothesis for the question. Keep in mind that:

Biology 05LA Fall Quarter 2013

Lab 4B page 5

a) You must learn as much as possible about what is known about related aspects of the question before you can formulate a hypothesis. b) Hypotheses should meet the criteria in item 3; a - d (page 2). c) Hypotheses should be expressed in the most concise and focused manner possible 2) Supporting argument for your hypothesis: Here you should write a concise, descriptive argument (entirely in your own words) in support of your hypothesis. This must be presented in a logical progression from general to specific - do not to assume that the reader is an expert in the field. You must also state the source of the facts used in your argument so that your TA can look these up. (e.g. Bio 05LA Lab Manual Lab # __, UCR, Summer 12 ed., pp.__-__, or Campbell Biology 9th ed. text, pp. __-__ ). Not citing the source of your facts will be considered plagiarism and will be penalized severely. Further, it is not acceptable to use direct quotes of factual information from the lab manual or the Campbell text in your argument. As stated above, this information must be presented in your own words. Two final comments: First, summary statements in the text or elsewhere are not facts and should be avoided. Second, because much misinformation is present on the WWW, citations from the internet will not be accepted. 3) Experimental strategy: What is needed here is a brief statement describing how the experimental approach to be used will relate to your hypothetical solution. What you need to convey here is the relationship between the experimental data that will be collected and the question you are attempting to answer. For example: a) How does the rate of color change in the reactions run in the enzyme lab relate to the activity of the alkaline phosphatase? b) How does a change in gas volume within the experimental tubes used in the fermentation and respiration experiments relate to the metabolic rate of the organisms in the tube? 4) Predictions: Remember that: predictions are best presented in an if/then form; if my hypothesis (here you need to actually state your hypothesis) is supportable and the experiment is done, then I should get the predicted result (here you need to actually state your prediction). Keep in mind that the prediction should relate to the actual experimental data that will be collected and not expressed in the more general terms used in your hypothesis. Completion of the write-up. The following items are required for the completion of the write-up. These should be presented in the lab notebook, but will not be graded. These items need to be prepared separately for each question. 1) Results. Begin by preparing the required graphs (if any) and then doing the required calculations. Next prepare a simple table of your processed data (e.g. the different rates for the different experimental conditions). Once the table is prepared, you then need to provide a brief statement describing what the table shows. Do not make interpretations of your results at this time. 2) Discussion. For each question you need to do the following. First, briefly and clearly state how well your predictions and results matched (be sure to mention the data that supports your claim). In the case of a good match, conclude with a simple declarative statement of what was learned this should be expressed in the terms of your hypothesis. In the case of a poor match, describe what you think went wrong and propose the specific changes that you think would improve your results were you to perform the experiment again. These changes may be to the manner in which you performed the experiment or to your original hypotheses. * * *

Biology 05LA Fall Quarter 2013

Lab 4B page 6

Submission of introductions: Your introductions must be submitted in two formats. 1) The first is as a single computer file containing both questions uploaded through Safe Assign, a program designed to check your assignment for plagiarism. You will find the link on your TAs iLearn site under Course Materials. The assignment is due by midnight the night before your lab and you have only one chance to submit the file. Do not procrastinate! 2) The second is as a printed copy of the file. This is the version due in lab. You must submit both versions for full credit. The questions that will be addressed by the experimental labs this quarter are listed below or within the relevant lab exercise. The Enzyme Lab. 1) How will increasing substrate concentration affect the rate (v0) of an enzymatic reaction if the enzyme concentration is held constant for the different trials? 2) How will altering pH away from the optimal pH affect the rate (v0) an enzymatic reaction? The Fermentation and Respiration Lab these will be presented with the lab exercise.

Learning Goals/Desired Outcomes for Lab 4

1) Be able to define and differentiate between the following pairs of terms: a) inductive reasoning deductive reasoning. b) hypothesis prediction. (an educated guess will not be considered as an adequate definition of a hypothesis!) c) experimental protocol experimental strategy. 2) Be able to explain why gaining a thorough knowledge of the known facts related to a particular question is a vital prerequisite to proposing a hypothesis for that question.

Biology 05LA Fall Quarter 2013

Lab 4B page 7

Biol 5LA Lab 4 In-class Worksheet

Name __________________________

1. Write a hypothesis for each of the following questions: (1/2 point each) a. How will an increase in temperature influence the rate of diffusion of that solute down its concentration gradient? b. How will the molecular mass of a solute molecule influence the rate of diffusion of that solute down its concentration gradient? c. How will placing a cheek cell in a hypotonic environment affect the cell? d. If two solutions having differing osmolarities are separated by a differentially selective membrane, in which direction will the solvent flow?

2. Name three supporting facts you should include in the argument to support your hypothesis for question 1a. (1 point)

3. What would be the experimental strategy for question 1a? (1 point)

4. Write a prediction for the hypotheses you generated above for questions 1a and 1c. (1/2 point each)

Biology 05LA Fall Quarter 2013

Lab 4B page 8

Some thoughts about the preparation of the two introductions you will be writing this quarter with particular emphasis on the Enzyme lab.
SUPPORTING FACTS. The biggest problem here is that many students make false assumptions about who is the audience for this document. They think they are writing this for their TA or the course faculty. This is definitely not the case. The correct target for this writing should be for someone who is not a scientist, but who is someone you want to enlighten about the topic. This means that you have to be very systematic about the way you develop the relevant scientific theory in support of your hypothesis and also to be careful to define all of the terminology used. In short, you want to prepare a document that any intelligent and curious person could pick up and understand. This requires that your argument be organized in a logical progression from general to specific. This can be challenging, but do-able for the science student. Start by carefully reading the questions so that you know what is being asked. Continue by reading the entire exercise in the lab manual. This will further clarify the question. Now that you have an idea about the question, it is time to learn about what is known about the subject of the question. To this end you must read your text, your lab manual, and your lecture notes for information pertaining to the question. When you come up with a piece of factual information that you think is relevant, write this down on a separate note card along with where you got it. Now you can FORMULATE A HYPOTHESIS based upon the facts you have collected this should not be too difficult for this lab. Hypotheses should be presented in the language of the question. You are now ready to assemble your argument for your hypothesis. This is where the note cards come in. These can be ordered in many different ways until you arrive at a progression that makes sense to you. At this time it can be seen what facts might be missing or what definitions might be necessary. These can then be inserted where necessary. The last bit of advice here is to put this aside for a while. When you come back to it, any remaining problems should be evident and easily corrected. Supporting Facts for Question 1 Define the term enzyme and each component of an enzymatic reaction. Define and describe the catalytic cycle of enzymatic reactions. Establish the active site as the part of an enzyme that binds substrate. Evaluate each step in the catalytic cycle to determine which step of the cycle is rate-limiting. Establish how the variable in question 1 could influence the rate limiting step determined above. Supporting Facts for Question 2. These will be much the same as for question 1. The difference lies in how the variable for question 2 influences the rate limiting step. Here you will probably need to broaden your fact search in the text. EXPERIMENTAL STRATEGY (ES) FOR BOTH QUESTIONS. Remember that ES is intended to make the connection between the actual data to be collected and your hypothesis. As stated in the lab manual, the key concern for the enzyme lab ES is: How does the rate of color change in the reactions run in the enzyme lab relate to the activity of the alkaline phosphatase? So, for this lab you will need to: Establish the basis for the color change. Describe how the color change will be monitored. Relate how the data collected is processed in order to come up with a value for reaction rate (v0)

While this sounds like a lot of work, the ES prepared for the first question is identical to that for the second question!!