biology 150 student lab manual 2010.pdf

Cell and Molecular Biology Laboratory Manual Third Edition

Exercise 1. Basic Instrumentation and Techniques It is important for a cell and molecular biology student to be familiar with the tools and equipment in the lab. Knowledge of proper care of the instruments will ensure their longevity, and knowledge of their proper use will aid in getting positive results when carrying out experiments. This exercise introduces the student to the cell and molecular biology laboratory. Objectives: At the end of this activity the student should be able to:

1. be familiar with the facilities used in cell and molecular biology; 2. prepare and organize simple setups needed for basic applications in cell biology; 3. operate equipment that are useful in analyzing cellular molecular components;

and 4. measure parameters that are important in understanding the cytological effects of

physical and chemical factors. Methodology

1. Take note of the following equipment and instruments whose usage you need to be familiar with in the conduct of cell and molecular biology laboratory protocols.

a. Bio Ref b. UV/Vis Spectrophotometer c. Photo Documentation System d. Micropipettors (0.5-10µl, 2-10µl, 20-100µl, 100-1000µl ) e. Thermal Cycler (MyCycler PCR Machine) f. Gel Electrophoresis System

i. Horizontal gel electrophoresis ii. Vertical gel electrophoresis iii. Power supply iv. Fast Blast Staining Facility

g. Shaker/Agitator h. Vortex mixer i. Microcentrifuge j. Water bath k. Distilling apparatus l. Autoclave/Sterilizer m. Ice shaver n. Parafilm

2. A battery of exercises will be conducted by your instructor to equip you with basic

skills necessary in conducting research in cell and molecular biology. a. Proper use of micropipettors b. Assembly of a gel electrophoresis system c. Spectrophotometry


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Your Task: Research on the descriptions and uses of the equipment and instruments in Part 1 of this exercise. Pay special attention to their function. Submit a 10- to 15-page report to your teacher. Include answers to the following in your report:

1. How is a spectrophotometer used to determine DNA concentration and purity? 2. The speed of a microcentrifuge may be measured in rpm or x g. How do you

convert between these two units? 3. In PCR, what is the relationship between the DNA template’s melting point and

the annealing temperature? Explain your answer briefly.


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Exercise 2. Nucleic Acids

The polymerase chain reaction (PCR), gel electrophoresis and restriction enzyme digestion are standard protocols in molecular biology. Data from these often serve as the basis for quantitative analyses, from which insight on the workings of the natural world may be gleaned. These tools are applied in a wide variety of fields such as population genetics, forensics, and the detection of genetically-modified organisms. Objectives: At the end of this activity the student should be able to:

1. isolate nucleic acids (DNA and RNA) from selected biologically important sources;

2. apply basic protocols in nucleic acid analysis such as polymerase chain reaction (PCR), agarose gel electrophoresis (AGE) and DNA fingerprinting;

3. conduct proper documentation and reporting of scientific findings; 4. understand the ethical issues in the applications of nucleic acid analysis; and 5. practice ethical behavior in the conduct of nucleic acid analysis procedures.

2A. Extraction of Eukaryotic DNA Materials Needed

Item/s Quantity Item/s Quantity cheek cells glass vials 36 lysis buffer A (40ml) 1 silver caps 36 lysis buffer B (40ml) 1 plastic plugs 36 protease (1.3ml) 1 waxed string 36 5M sodium chloride (salt), 5ml 1 super glue gel 2 sterile water, 2.5 ml 1 91% isopropyl alcohol or 95% EtOH 1 5-ml round bottom tubes 50 water bath with thermometer 1 clear micro test tubes 60 colored micro test tubes 60 screwcap tubes (assorted color caps) 40 disposable plastic transfer pipets 50 foam micro test tube holders 10 cytology brushes 80 parafilm laboratory film 1


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Methodology Pre-Lab Preparations Each workstation should have the following: Items Units 15-ml tubes containing 3 ml of water 2 micro test tube labeled "prot" containing 1 1.25 ml of protease + salt 15-ml tube labeled "lysis" containing 10 ml lysis 1 buffer disposable plastic transfer pipettes 3 foam micro test tube holder 1 permanent marker 1 disposable paper cup for holding 15-ml tubes 1 waste vessel 1 1. Assign one or two representative/s from your group. 2. Secure 250 ml 95% ethanol from the stockroom and store it in the refrigerator. 3. Make sure that the sodium chloride to be used in this experiment is not iodized. 4. The use of E. coli in this experiment is optional. Inquire about how to acquire non-pathogenic E. coli from your instructor.


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Laboratory Proper


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Your Task: Prepare a 3- to 5-page formal report, explaining the rationale for each step in this exercise. Explain the results you obtained. Your report should have an Abstract, Title, Introduction, Methodology, Results and Discussion, and Conclusion. Make sure that the problem and hypothesis are stated in the Introduction. Include answers to the following in your report: 1. What is the reason for using 95% ice-cold ethanol in the extraction procedure? 2. What color of the precipitated DNA should you expect? Explain your answer. Reading assignment: Steiner JJ, CJ Poklemba, RG Fjellstrom & LF Elliott. 1995. A rapid one-tube genomic

DNA extraction process for PCR and RAPD analyses. Nucl. Acids Res. 23:2569-2670.


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2B. DNA Fingerprinting Materials Needed

Item/s Quantity Item/s Quantity HindIII DNA size standard, 100µl 1 horizontal gel

electrophoresis chambers 2

DNA samples, lyophilized, 60 µg each micropipets Crime scene sample 1 2-20µl 1-8

Suspect samples 5 20-200µl 1

EcoRI/Pstl restriction enzyme mix 1 100-1000µl 1

Lyophilized, 3000 units pipet tips sample loading buffer, 5x, 1ml 1 2-20µl 1 box

electrophoresis buffer, 50x TAE, 100 ml 1 20-200µl 1 box

sterile water, 2.5 ml 1 100-1000µl 1 box

agarose powder, 5 g 1 power supply Fast Blast DNA stain, 500x, 100 ml 1 water bath colored micro test tubes, 1.5 ml 80 mini centrifuge foam micro test tube holders 8 gel support film gel staining trays 4 hot plate Methodology: Pre-Lab Preparations 1. Rehydrate DNA/ buffer samples and restriction enzymes. a. Add 200 µl of sterile water to the vials containing the DNA/buffer samples. b. Allow to rehydrate for 5 min at room temperature. Wait until everything is dissolved. c. Incubate the DNA/buffer samples in a 37oC water bath. 2. Rehydrate and aliquot restriction enzymes. a. Add 750 µl of sterile water to EcoRI/Pstl enzyme mix. b. Allow rehydration to take place on ice for 5 min. This enzyme mix preparation will

be good within 12 hours. c. Aliquot 80 µl of enzyme mix into a 1.5-ml microtube and label that tube "ENZ". 3. Preparation for gel electrophoresis. a. Weigh an appropriate amount (in grams) of agarose gel powder enough for a

1%(w/v) or 2%(w/v) agarose gel. b. Prepare 1X TAE buffer from the 50X stock solution. c. Melt the agarose gel powder in 1X TAE buffer*. d. Secure the gel casting tray with masking tape. Pour the gel in the gel casting tray,

put the gel comb in place, and allow the gel to polymerize. e. Approximately 275 ml of 1XTAE will be needed as running buffer. 4. Prepare 100X Fast Blast stain from the 500X stock solution. Use distilled water to dilute the dye in an Erlenmeyer flask. The dye may be stored at room temperature until it is ready for use. 5. Maintain a water bath temperature of 45 to 55oC for DNA visualization procedures.


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*Caution: Wear protective gloves, goggles and lab gown when preparing agarose gels to prevent accidents such as skin burns. Laboratory Proper


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3. Determine the culprit. a. Match the crime scene DNA with the suspects' DNA. b. Construct a standard curve using DNA size standards. c. Determine the size of unknown fragments in the DNA samples. d. Draw a possible conclusion on who the culprit was, based on the profiles in the gel. e. Note your findings in your logbook. 4. Document your findings using photodocumentation equipment (camera or scanner). 5. Place the gel in a plastic container with your group identification and store the gel in

the cell biology lab refrigerator.

Your Task: Prepare a 3- to 5-page formal report, explaining the rationale for each step in this exercise. Explain the results you obtained. Your report should have an Abstract, Title, Introduction, Methodology, Results and Discussion, and Conclusion. Make sure that the problem and hypothesis are stated in the Introduction.


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2C. Restriction Fragment Length Polymorphism Materials Needed

Item/s Quantity Item/s Quantity HindIII restriction enzyme 1 horizontal gel electrophoresis

apparatus 2

Pstl restriction enzyme 1 micropipets EcoRI restriction enzyme 1 2-20µl 4-8 restriction buffer 1 20-200µl 1 λ DNA, uncut 1 pipet tips 1 bag DNA size standard 1 2-200µl sample loading buffer, 5x, 1 ml 1 power supply 2 agarose powder, 5 g 1 water bath 1 electrophoresis buffer, 50x TAE, 100 ml

1 rocking platform

agarose powder, 5 g 1 gel support film Fast Blast DNA stain, 500x, 100 ml

1 hot plate

micro test tubes 60 foam micro test tube holders 8 gel staining trays Methodology Pre-Lab Preparations 1. Aliquot 3 µl of each restriction enzyme into three clear micro test tubes. Label the

tubes HindIII, Pstl and EcoRI respectively. Make sure that the micro test tubes are maintained on crushed ice.

2. Aliquot 30 µl of restriction buffer into two clear micro test tubes. Label the tube "res". Keep the tube on ice.

3. Aliquot 25 µl λ DNA into two clear micro test tubes. Label the tube "λ". Maintain the tubes on ice as well.

4. Aliquot 10 µl sample loading dye into two clear micro test tubes. 5. Store the enzymes, buffer, the λ DNA and sample loading dye in the cell biology

refrigerator. 6. Prepare 1X TAE buffer that will be enough for a 1%(w/v) or 2%(w/v) agarose gel

and electrophoresis chamber if necessary. 7. Prepare the agarose gel for the electrophoresis.


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Laboratory Proper


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Your Task: Prepare a 3- to 5-page formal report, explaining the rationale for each step in this exercise. Explain the results you obtained. Your report should have a Title, Introduction, Methodology, Results and Discussion, and Conclusion. Make sure that the problem and hypothesis are stated in the Introduction.


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2D. Polymerase Chain Reaction Materials Needed

Item/s Quantity Item/s Quantity positive Controls micro test tubes 60 homozygous (+/+) 1 PCR tubes 0.2 ml 50 heterozygous (+/-) 1 screwcap tubes 1.5 ml 50 homozygous (-/-) 1 capless PCR tube adaptors

1.5 ml 50

PCR Master Mix 1 agarose powder 5 g 1 PCR Primer Pair 1 electropheresis buffer 1 DNA molecular mass ruler 1 Fast Blast DNA stain 1 InstaGene™ DNA extraction matrix

1 foam micro test tube holders 8

xylene cyanol DNA loading dye 1 gel staining trays 4 Methodology Pre-Lab Preparations 1. Pipet 200 µl of Instagene™ matrix into four screwcap tubes. Make sure that the

contents of the bottle have been gently mixed or vortexed prior to pipetting. 2. Prepare 50 ml 0.9% saline solution by adding 4.5g non-iodinated table salt to 500 ml drinking water. Give 10 ml of this to the class volunteer. 3. Pipet 34.375 µl (x4) of Master Mix to a yellow micro test tube labeled "complete mix". To this, add 0.6875 µl (x4) of the Primer Mix. The volume of this complete master mix

should total 140.25 µl. Vortex or flick the tube with your fingers. 4. Aliquot 20 µl of the complete master mix to each of four PCR tubes. 5. Prepare 1%(w/v) or 2%(w/v) agarose gel and appropriate volume of 1X TAE buffer. 6. Set the water bath to 100oC. Laboratory Proper (protocol to be provided by your instructor)


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Your Task: Prepare a 3- to 5-page formal report, explaining the rationale for each step in this exercise. Explain the results you obtained. Your report should have an Abstract, Title, Introduction, Methodology, Results and Discussion, and Conclusion. Make sure that the problem and hypothesis are stated in the Introduction. Include answers to the following in your report: 1. What are ALU repeat sequences? What is their evolutionary significance? 2. Explain briefly why PCR primers (forward and reverse) should be prepared in equimolar concentrations for a standard PCR technique. 3. What are the characteristics of a good PCR primer? Give three.


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2E. Organismic Genetics Some microorganisms such as E. coli are invaluable tools in molecular biology. Often, they serve as vectors in transformation experiments. Knowing how to manage them is an essential basic skill not only in molecular biology, but in related fields. This exercise also serves as the student’s first foray into genetic engineering. Objectives: At the end of this activity the student should be able to:

1. be familiar with some microorganisms used in cell and molecular biology; 2. prepare basic media needed for bacterial growth; 3. apply aseptic techniques in maintaining bacterial cultures; and 4. perform standardization procedures necessary for establishing baseline bacterial

counts. Materials Needed

Item/s Quantity Item/s Quantity plasmid (pGLO), lyophilized 1 inoculation loops, sterile 80 E. coli strain HB101 K-12, lyophilized

1 micro test tubes, 2.0 ml, sterile, color coded

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LB nutrient broth, sterile 1 foam micro test tube holders 8 LB nutrient agar powder 1 disposable plastic transfer pipets 50 Ampicillin, lyophilized 1 Arabinose, lyophilized 1 sterile transformation solution (CaCl2)

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Petri dishes, 60 mm, sterile 40 Methodology Pre-Laboratory Preparations 1. Prepare the nutrient agar three days before the experiment. This nutrient agar is

autoclave free. a. Fill a 1-L Erlenmeyer flask with 500 ml distilled water. b. Pour in the entire contents of the LB nutrient agar packet.

c. Dissolve the agar by swirling and heat to boiling. d. Repeat the above procedure until all agar is dissolved.

e. Wait until the agar cools slightly so that it will be comfortable to hold the outside of the flask. f. Do not let the agar cool too much that it will solidify. 2. Prepare the arabinose and ampicillin a. Add 3 ml of transformation solution to the vial containing lyophilized

arabinose. Use a fresh sterile pipet. It normally takes about 10 min for the arabinose to dissolve.

b. Hydrate the arabinose by following the procedure for the hydration of ampicillin.


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c. Note that the ampicillin and arabinose can be destroyed at temperatures that exceed 50oC.

3. Mark the plates. a. Label 16 plates "LB".

b. Label 16 plates "LB/amp". c. Label 8 plates "LB/amp/ara".

4. Pour the LB nutrient agar into the plates. Stack the plates 4 to 8 high. Starting with the

bottom plate, lift the lid straight up and to the side with one hand, and pour the LB nutrient agar with the other. Fill each plate about one-third to one-half (approx. 12 ml) with agar.

a. Pour the LB nutrient agar into the 16 plates labeled LB. b. To the remaining LB nutrient agar, add the hydrated ampicillin. c. Briefly swirl the flask to mix its contents. d. Pour into the 16 plates labeled LB/amp e. To the remaining LB nutrient agar with ampicillin, add the hydrated arabinose.

f. Briefly swirl the flask to mix its contents. g. Pour into the 8 plates labeled LB/amp/ara. 5. Allow the plates to cure. They should stay at room temperature for two days followed

by refrigeration.

a. After curing, the plates can be stacked twenty high and a plastic sleeve bag slipped down over them.

b. Invert the stack and secure the bag with tape. c. Store the plates inverted in the refrigerator.

6. Rehydrate bacteria and streak starter plates. a. Obtain lyophilized E. coli strain HB101 K-12.

b. Add 250 µl of transformation solution to the vial with lyophilized E. coli HB101. c. Allow the suspension to stand for 5 min. Then shake to mix. d. Store the rehydrated bacteria in the refrigerator within 24 hrs for no longer than 3 days.

e. Streak the bacteria on prepared agar plates, observing aseptic techniques. Streaking should be done sequentially in four quadrants. As you finish streaking one quadrant, rotate the plate to the next and repeat the streaking. f. Incubate the plates at 37oC overnight or for 2-3 days at room temperature. These can be used for transformation within 24-36 hours.

7. Prepare the plasmid by adding 250 µl transformation solution in the vial containing the

pGLO plasmid followed by refrigeration.


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Laboratory Proper


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13. Analyze results by comparing the appearance of the bacterial samples under UV light. Tabulate your observations:

EXPERIMENTAL PLATES CONTROL PLATES

+pGLO LB/amp

+pGLO LB/amp/ara

-pGLO LB/amp

-pGLO LB

3. Maintain the samples by making a stock culture for future use.

Your Task: Prepare a 3- to 5-page formal report, explaining the rationale for each step in this exercise. Explain the results you obtained. Your report should have an Abstract, Title, Introduction, Methodology, Results and Discussion, and Conclusion. Make sure that the problem and hypothesis are stated in the Introduction. Include answers to the following in your report: 1. What are plasmids? Give two possible advantages and disadvantages of plasmids (with or without human intervention). 2. Why is arabinose important in this experiment? 3. What are the possible uses of GFP? Why transfer the capability of GFP synthesis to E. coli O157:H7 strain?


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Exercise 3. Proteins

Being biologically all-important, proteins are a much-studied group of diverse molecules. Various isolation and characterization techniques have been developed for proteins, some of which will be performed in this exercise. Such steps are imperative in unlocking the properties of proteins which allow them to perform their biological roles, such as as enzymes, hormones or structural components of cells. We may also exploit these molecules in food production, drug design, and other areas, to benefit mankind. Objectives: At the end of this activity the student should be able to:

1. isolate proteins from selected biological sources; 2. apply basic protocols in analyzing proteins such as Bradford and Lowry method,

polyacrylamide gel electrophoresis (PAGE), basic column chromatography and enzyme linked immunosorbent assay (ELISA);

3. conduct proper documentation of scientific findings; 4. understand the ethical issues in the applications of protein analysis; and 5. practice ethical behavior in the conduct of protein analysis procedures.

3A. Protein Isolation and Quantitation Materials Needed

Item/s Quantity 1x Bradford dye reagent (12 ml) 1 microtubes with protein standards 7 test milk samples 2 1x PBS (500 µl) 1 microtubes for making dilutions 4 100–1,000 µl adjustable-volume micropipet 1 100–1,000 µl pipet tips 1 box 2–20 µl pipet tips 1 box cuvettes (or test tube substitutes) 10 milk carton with nutrition information 1 Parafilm (small pieces to seal cuvettes) 10


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Methodology

Use a wavelength of 595 nm.


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Your Task: Prepare a 3- to 5-page formal report, explaining the rationale for each step in this exercise. Explain the results you obtained. Your report should have an Abstract, Title, Introduction, Methodology, Results and Discussion, and Conclusion. Make sure that the problem and hypothesis are stated in the Introduction. Include answers to the following in your report: 1. What is the principle behind the use of Bradford reagent in this exercise? Why use a wavelength of 595 nm? 2. Did your findings about the protein content of your samples match with the product information provided? If no, can you explain why. 3. What is the significance of using a control in this activity?


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3B. Protein Fingerprinting Materials Needed

Item/s Quantity Item/s Quantity pipet tips for gel loading 1 rack Actin and Myosin Standards

500 µg 1

micro test tubes 1.5 ml 120 foam micro test tube holders 8 screwcap micro test tubes 1.5 ml 200 gel staining trays 4 disposable plastic transfer pipets 1 ml

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Laemli sample buffer 30 ml 1 Kaleidoscope prestained standards 500 µl

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electrophoresis buffer 10x TRIS/glycine/SDS, 1L

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Biosafe Coomasie Stain 1L 1 Methodology

1. Extract fish muscle proteins: a. Put appropriate amounts of fish muscle and Laemli sample buffer in a

micro test tube. b. Gently flick the tube to agitate the sample.

2. Incubate samples for 5 minutes at room temperature. 3. Pour extracted protein samples into screwcap tubes. 4. Heat protein samples at 950C for 5 minutes. 5. Load samples on a polyacrylamide gel and electrophorese at 200V for 30

minutes. Load the protein standards in the first or second lane. 6. After the run, remove the gel from the chamber and stain it with Bio-Safe

Coomasie stain and destain with water. 7. Analyze the results by comparing the sample profiles to that of the protein

standards. Determine which fish species are more closely related and which are not. Construct a dendrogam to illustrate your findings.


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Your Task: Prepare a 3- to 5-page formal report, explaining the rationale for each step in this exercise. Explain the results you obtained. Your report should have an Abstract, Title, Introduction, Methodology, Results and Discussion, and Conclusion. Make sure that the problem and hypothesis are stated in the Introduction. Include answers to the following in your report: 1. What is the evolutionary significance of fish muscle proteins? 2. Is it possible to further purify the proteins resolved using PAGE? How? 3. What is the advantage of using prestained protein standards in this technique?


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3C. Hydrophobic Interaction Chromatography Materials Needed

Item/s Quantity Item/s Quantity Ampicillin, lyophilized 1 HIC chromatography columns 8 Arabinose, lyophilized 1 inoculation loops, sterile 20 Lysozyme, lyophilized 1 disposable plastic transfer pipets 40 LB nutrient broth tablet 1 micro test tubes, 2 ml, clear 30 binding buffer 1 cell culture tubes, 15 ml, sterile 25 column equilibration buffer 1 sample collection tubes, 5 ml 25 column wash buffer 1 foam micro test tube holders 8 elution buffer 1 Methodology Pre-Laboratory Preparations Prepare Ampicillin and Arabinose Solutions 1. Using a sterile pipette, add 3 ml TE solution directly to the vial containing ampicillin. 2. Using another sterile pipette, add 3 ml TE solution to rehydrate the arabinose. 3. Mix or vortex the vials and swirl to facilitate rehydration. Prepare Liquid Nutrient Media 1. Put 55 ml distilled water in a 250 ml Erlenmeyer flask and heat to boiling. 2. Add into it 1 LB tablet. Allow the tablet to soak for 20 min, followed by heating until

boiling is reached once again. Allow the flask to cool a bit then swirl the contents carefully.

3. Heat and swirl again until the entire tablet is dissolved. 4. When the flask's temperature is below 50oC add into it 0.5 ml of arabinose and 0.5 ml of ampicillin. Mix the components by swirling. 5. Aliquot 2 ml of the liquid media into 16 to 25 culture tubes. Laboratory Proper (protocol to be provided by your instructor)


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Your Task: Prepare a 3- to 5-page formal report, explaining the rationale for each step in this exercise. Explain the results you obtained. Your report should have an Abstract, Title, Introduction, Methodology, Results and Discussion, and Conclusion. Make sure that the problem and hypothesis are stated in the Introduction. Include answers to the following in your report: 1. How can a simple technique like size-exclusion column chromatography exhibit

specificity in isolating a protein of interest? Explain the role of the buffers used in eluting your protein.

2. Why do you need to purify your protein given that it already exhibits biological activity even in crude state?


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3D. Enzyme-Linked Immunosorbent Assay (ELISA) Materials Needed

Item/s Quantity Item/s Quantity antigen (chicken צ-globulin) 1 microplates with 12-well strips 3 primary antibody (rabit anti-chicken polyclonal antibody)

1 yellow micro test tubes, 2.0 ml 60

secondary antibody (goat anti-body conjugated to horse radish peroxidase, or HPP)

1 colored micro test tubes, 2 ml 75

HRP enzyme substrate, 25 ml 1 10x PBS, 100 ml 1 10% Tween 20, 5 ml 1 disposable plastic transfer pipets 80 empty 30-ml bottles and caps 3 Methodology Pre-Lab Preparations Prepare the buffers. 1. Prepare at least 100 ml 1x phosphate buffered saline (PBS) from 10x PBS. 2. Prepare a wash buffer by adding 90 ml 10x PBS to 805 ml distilled water and add 4.5 ml to 5 ml 10% Tween 20 to make a final volume of 900 ml. Prepare antigen, primary antibody and secondary antibody stock solutions. 1. Add 0.5 ml 1x PBS to a vial of the antigen. 2. Add 0.5 ml 1x PBS to a vial of primary antibody. 3. Add 0.5 ml 1x PBS to a vial of secondary antibody. Prepare working solutions. 1. Label one 30-ml bottle each for the antigen, primary antibody and secondary antibody. 2. Transfer 7.5 ml 1x PBS to a 30-ml bottle labeled "antigen". To it add 150 µl of 50x antigen stock solution. 3. Transfer 24.5 ml wash buffer to a 30-ml bottle labeled "primary antibody". To it add 0.5 ml 50x primary antibody. 4. Repeat the previous procedure for the secondary antibody. Prepare the secondary antibody less than 24 hours before the experiment. Dispense the reagents to group representatives. 1. Transfer 0.5 ml 1x antigen to a violet micro tube. This is your positive control. Label it "+" 2. Transfer 0.5 ml 1x PBS to a blue micro tube. This is your negative control. Label it "-" 3. Transfer 1.5 ml 1x primary antibody to a green micro tube. Label it "PA".


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4. Transfer 1.5 ml 1x secondary antibody to an orange micro tube. Label it "SA". 5. Transfer 1.5 ml HRP enzyme substrate to a brown tube. Label it "SUB". 6. Reserve a yellow micro tube for the infected sample. Laboratory Proper

strip.


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Your Task: Prepare a 3- to 5-page formal report, explaining the rationale for each step in this exercise. Explain the results you obtained. Your report should have an Abstract, Title, Introduction, Methodology, Results and Discussion, and Conclusion. Make sure that the problem and hypothesis are stated in the Introduction. Include the answers to the following in your report: 1. What is the advantage of using a secondary antibody instead of only a primary

one? 2. Were false positives or negatives encountered in this activity? How could this affect the final analysis of results? How can such errors be prevented?


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Exercise 4. Bioinformatics Efficiency and specificity in identification, verification and annotation of novel biomolecules like proteins and nucleic acids targeted for a special biomedical and industrial purposes can now be achieved in silico through the use of computer programs that can either be standalone or online. Simple yet robust, biocomputing technology using updated information shared by research-based computer networks through online internet access to biological databases worldwide has become widespread. 4A. Using Biological Databases

1. Quick Sequence Searches a. Go to http://www.ebi.ac.uk/fasta33/index.html. Do a protein-protein FastA search with the following sequence (which you may cut and paste) and use the default parameters:

>WHOAMI

STKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQSGVGAL

FNGINVLQAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSE

YEYPVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSM

TAPTGSKPSFPDGMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQ

VFLQPLNPQYPMIPCNESCSVVGKVIASQWPEETFG

Which protein can be assigned to this sequence? b. Run a different search of the following sequence:

MRFPELEELKNRRTLKWTRFPEDVLPLWVAESDFGTCPQLKEAMADAVER

EVFGYPPDATGLNDALTGFYERRYGFGPNPESVFAIPDVVRGLKLAIEHF

TKPGSAIIVPLPAYPPFIELPKVTGRQAIYIDAHEYDLKEIEKAFADGAG

SLLFCNPHNPLGTVFSEEYIRELTDIAAKYDARIIVDEIHAPLVYEGTHV

VAAGVSENAANTCITITATSKAWNTAGLKCAQIFFSNEADVKAWKNLSDI

TRDGVSILGLIAAETVYNEGEEFLDESIQILKDNRDFAAAELEKLGVKVY

APDSTYLMWLDFAGTKIEEAPSKILREEGKVMLNDGAAFGGFTTCARLNF

ACSRETLEEGLRRIASVL

Identify the protein with the above sequence. c. Do an online BLAST search at http://www.ebi.ac.uk/blastall/. Use the sequences above with the default parameters. Are the proteins the same as those identified in the FastA search?

2. Use of sequences to determine phylogenetic relationships Go to http://www.expasy.ch/cgi-bin/sprot-search-ful and type in the keywords horse pancreatic ribonuclease followed by the ENTER key. Select RNAS1_HORSE_, SEQUENCE and then FASTA format. This sequence may be copied and pasted onto other programs.


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Retrieve sequences of minke whale (Balaenoptera acutorostrata) and red kangaroo (Macrapus rufus) ribonucleases using the above procedure. Copy and paste the retrieved sequences into the multiple-sequence alignment program CLUSTAL-W at http://www.ebi.ac.uk/clustalw/ and run that program.

3. Exploration of a Database PubMed is the public and freely available version of Medline, the most comprehensive database of primary scientific literature in the biomedical area. Maintained by the U.S. National Library of Medicine (physically located on the campus of the National Institutes of Health in Bethesda, Maryland), PubMed is the brainchild of the NCBI (National Center for Biotechnology Information), a world-leading bioinformatics center. The nucleotide sequence database GenBank and the immensely popular BLAST software are two other famous productions of NCBI. Searching PubMed

1. Go to www.pubmed.nl 2. Type in dUTPase in the For window, and click the Go button. Is the database

search case-sensitive? 3. For any entry in the Results list, click the associated author names. 4. Save what you like to your hard drive by choosing your browser’s File � Save As

option. Alternatively, you can transform the display into a simpler (printer-friendly) format by first clicking the Text button on the right of the line with Display, and then choosing File � Save As.

Saving Multiple Summaries

When your search yields many references, the best move is to start scanning a few pages and select the most promising papers for future use by checking the corresponding boxes. To move through the Results pages, just use the Select page button in the top right to select the next page.

1. Choose Abstract from the pull-down menu to the right of the Display button. 2. If you want to print this display as is, choose File � Print from your browser

menu. 3. If you want to print this display in a more printer-friendly format, first click the

Text button (on the same menu bar as the Display button) to display this information in a non-HTML test format and then choose File � Print from your browser menu.

4. If you’d like to save the file in the format of your choice, choose File � Save As from your browser menu, enter a new filename for the file, and then choose the file format you want to save to.


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Searching PubMed using author’s names

1. Point your browser to www.pubmed.nl 2. Choose PubMed from the drop-down menu for the Search window. Type

Abergel – the name of your prospective author – in the For window, and then click the Go button.

3. Type dUTPase next to Abergel in the For window and click Go. 4. Click the underlined names of the authors to access the page containing a

summary of the paper. 5. Click one of the Free Text rectangles. Either link brings you directly to the

publisher’s site, where you have the option of reading an interactive online copy (HTML) or downloading reprints in pdf format, among other choices.

Searching PubMed using fields

1. Point your browser to www.pubmed.nl 2. Type dUTPase and Abergel in the For window, and then click the Go. 3. Click the small arrow of the pull-down menu, just to the right of the Display

window. 4. Select the Medline option. The Medline page appears. You will see the

internal structure of a Medline database record. The information is spread out over separate sections, called fields, each one preceded by a specific abbreviation – TI for title fields, AB for abstracts, AD for the laboratory address, AU for the authors, SO for the journal abbreviation, and so forth. This structure applies to all Medline records. Unfortunately, not all PubMed searches are as easy and productive as the one we used in the dUTPase example. It is possible to come up with an overwhelming number of hits, for example, if you formulate queries that contain common names (such as Smith) or use search terms (such as Down) that can occur in different contexts: titles (for example, Down syndrome), abstracts, author name, or can be part of an address (955 Down Street).

5. Query PubMed by restricting the search for each word of your query to a given field. Try entering three different queries – Down [AU], Down [AB], and Down [AD] into the For box at the PubMed site. How many hits for each search term did you get?

6. Type in the search term dUTPase [AB] Chicago [AD] and click Go. You will get a list of experts on dUTPase in Chicago.

7. Obtain the abstracts of the articles in the Results page. Searching PubMed using limits When you want to quickly find out the basics about a subject you know nothing about, it’s best not to read any single highly specialized article. A better bet is to stick with review articles, where one expert summarizes the state-of-the-art for you. Thus, we want to limit our PubMed search to recent review articles about dUTPase.

1. Point your browser to the PubMed site and type dUTPase in the For window. 2. Click the Limits button. The Limits screen appears. You now have plenty of fields

and attributes you can use for setting limits. You can go back later to this page and explore these various options.

3. Choose Review as the Type of Article and English as the Language (unless you


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are fluent in French). 4. Choose a suitable year for the Published in the Last box, such as 5 years. 5. Select Title/Abstract in the pull-down menu for Tag Terms. The default here is All

Fields. Choosing Title/Abstract restricts the occurrence of dUTPase in the Title or Abstract fields in Medline records.

6. Finally, click Go. Your (new and more concise) Results page appears. A few more tips about PubMed

• Quoted queries (for example, “down syndrome”) behave as a single word, and are a great way to improve the relevance of your search.

• Adding initials to proper names (for example, Abergel C) can greatly reduce the number of hits.

• Write down the PubMed Identifier (the number in the PMID field) of that interesting paper you just found. It can be very useful in any subsequent searches for related items, such as associated gene and protein sequences.

• Don’t forget to deselect the Limit box when starting a new search.

• Don’t put too much initial faith in a search that produces no results. Spelling mistakes, wrong field restrictions, or improper limits settings can all throw off an effective search.

• As a beginner researching anew subject, read through a couple of abstracts to enlarge your initial vocabulary and look for synonyms. For example, if you don’t know that some papers on dUTPase might use the term “dUTP pyrophosphatase” instead, you may miss out on some interesting papers.

• Try the Related Papers button to enlarge a search that isn’t giving you enough references.

Things you won’t find in PubMed

o Names ranking beyond the tenth place in the author’s list aren’t retrievable for older papers (before 1995). This is a significant problem for many pioneering articles on genomics.

o No papers recorded before 1965 are in PubMed. Don’t rely on PubMed to write an historical article on your field.

o Most references recorded before 1976 have no abstract. Don’t expect great results from PubMed searches involving them.


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4B. Molecular Visualization

RasMol: A Molecular Graphics Visualization Tool exerpts from the Manual for RasMol 2.7.1

RasMol is a molecular graphics program intended for the visualization of proteins, nucleic acids and small molecules. It can be downloaded for free from the Internet. To start RasMol under Microsoft Windows, double click on the RasMol icon in the program manager. Two windows will appear, the main graphics or canvas window with a black background and a command line or terminal window. At the top of the graphics window is the RasMol menu bar, on which the 'File', 'Display', 'Colors', 'Export' and 'Options' pull-down menus are found. The Main graphics window also has two scroll bars, one on the right and one at the bottom, that may be used to rotate the molecule interactively. The program reads in a molecular coordinate file (such as a PDB file) and interactively displays the molecule on the screen in a variety of representations and color schemes. Mouse Controls Here is a summary of RasMol's mouse click-and-drag controls. Action Buttons/Controls Rotate X, Y Left Translate X, Y Right Rotate Z Shift-Right Zoom Shift-Left Slab Plane Ctrl-Left Picking In order to identify a particular atom or bond being displayed, RasMol allows the user to 'pick' objects on the screen. The mouse is used to position the cursor over the appropriate item, and then any of the mouse buttons is depressed. Provided that the pointer is located close enough to a visible object, the program determines the identity of the nearest atom to the point identified. The program will display, in the terminal window, the atom's type, serial number, residue name and residue number. If the atom is a member of a named chain, the chain identifier is also displayed. Two examples of the output generated by selecting an atom follow: Atom: CA 349 Group: SER 70 Atom: O 526 Hetero: HOH 205 Chain: P The first line describes the alpha carbon of the serine-70 amino acid in a protein. The unique Protein Data Bank serial number for this atom is 349. The following line describes the oxygen atom in a water molecule attached to the P chain of the main


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molecule. The word 'Hetero' distinguishes heterogeneous molecules (such as cofactors) from the residues in the main molecule, noted by 'Group' Clicking the mouse on an atom can be used not only to identify it, but also to find the coordinates, the distances between two atoms, the bond angle defined by three atoms, the torsion angle defined by four atoms, to toggle labels on or off, or to specify the center of rotation.

Exercise. Interactive inspection of a molecule using Rasmol 1. Start Rasmol by double-clicking on its icon. 2. Open a PDB file by selecting File>Open from the drop-down menu bar of the graphics window. 3. Select the file "helix". You will see a graphical representation of a helix in the main window of Rasmol. How many turns does the helix have? 4. Move the molecule around by holding down the left mouse button while moving the mouse. 5. Try changing the appearance of the molecule by using the options on the "Display" button of the drop-down menu. You can choose from Backbone, Spacefill, Cartoon, Ribbon, Wireframe, etc. 6. Try changing the color of the molecule by using the options on the "Colours" button of the drop-down menu. Alternatively, you can type the following in the Command Line Window: select all <Return> color red <Return>. The molecule in the display window will appear red. You may also type in other colors aside from red. 7. In the Command Line Window, type set picking distance. Then using the mouse, click on any two atoms in the Graphics Window. The distance between these two atoms (in Angstroms) will be indicated in the Command Line Window. Click on another two atoms to find out the distance between them.

For more details on RasMol and other molecular visualization freeware, please visit: http://www.umass.edu/microbio/rasmol/


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Exercise 5. DNA Microarray

DNA microarray analysis is one of the newest tools in the fields of molecular biology and medicine. Scientists are using DNA microarrays in a host of experiments, including investigating changes in gene expression levels and identifying single nucleotide polymorphisms. This exercise is meant to introduce the student to the basics of this new technology.

Materials needed

Computer with internet connection Methodology Point your internet browser to http://learn.genetics.utah.edu/content/labs/microarray/ and follow the instructions therein. Here you will use a DNA microarray to investigate the differences between a healthy cell and a cancer cell.


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References

Books Ausubel FM, R Brent, R Kingston, D Moore, JG Seidman, JA Smith and K Struhl. Short

Protocols in Molecular Biology. Green Publishing Associates and John Wiley and Sons, New York, 1992

Claverie JM & C Notredame. Bioinformatics for Dummies. Wiley Publishing, Inc., New

York, 2003. Davis LG, MW Kuehl and JF Battey. Basic Methods in Molecular Biology 2nd Ed.

Appleton and Lange. Norwalk Connecticut. 1994. Lesk AM. Introduction to Bioinformatics. Oxford University Press, Oxford, 2002. Scientific Journals Hartmann K. 2003. Applied Bioinformatics: Quick Sequence Searches in Databases.

CUBIC lecture. Steiner et al. 1995. A rapid one-tube genomic DNA extraction process for PCR and

RAPD analyses. Nucl. Acids Res. 23: 2569-2570 Monographs Bio-Rad Biotechnology ExplorerTM Chromosome 16: PV92 PCR Informatics Kit

Bio-Rad Biotechnology ExplorerTM DNA Fingerprinting Kit

Bio-Rad Biotechnology ExplorerTM ELISA Immuno Explorer

Bio-Rad Biotechnology ExplorerTM Fast Blast DNA Stain

Bio-Rad Biotechnology ExplorerTM Genes in a Bottle Kit DNA Extraction Module

Bio-Rad Biotechnology ExplorerTM Green Flourescent Protein (GFP) Purification Kit Bio-Rad Biotechnology ExplorerTM pGLO Bacterial Transformation Kit. Bio-Rad Biotechnology ExplorerTM Protein Fingerprinting

Bio-Rad Biotechnology ExplorerTM Restriction Digestion and Analysis of Lambda DNA Kit

EDVOTEK® The Biotechnology Education Company® PCR-based Testing for Water Bacterial Contaminants


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Online Manual for RasMol 2.7.1. Available at http://www.bernstein-plus-

sons.com/software/RasMol_2.7.1/doc/rasmol.html DNA Microarray Virtual Lab. 24 May 2010. The University of Utah. 25 May 2010.

http://learn.genetics.utah.edu/content/labs/microarray/.

biology 150 student lab manual 2010.pdf

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