Biology 150 Student Lab Manual 2010

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
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.
2
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.
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
3
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.
4
Laboratory Proper
5
6
7
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.
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.
8
2B. DNA Fingerprinting
Materials Needed
HindIII DNA size standard, 100µl 1 horizontal gel 2
electrophoresis chambers
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:
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.
9
*Caution: Wear protective gloves, goggles and lab gown when

preparing agarose gels to prevent accidents such as
skin burns.
Laboratory Proper
10
11
12
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:
13
2C. Restriction Fragment Length Polymorphism
Materials Needed
HindIII restriction enzyme 1 horizontal gel electrophoresis 2
apparatus
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 1 rocking platform
TAE, 100 ml
agarose powder, 5 g 1 gel support film
Fast Blast DNA stain, 500x, 1 hot plate
100 ml
micro test tubes 60
foam micro test tube holders 8
gel staining trays
Methodology
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.
14
Laboratory Proper
15
16
17
Your Task:
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.
18
2D. Polymerase Chain Reaction
Materials Needed
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 50
1.5 ml
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 1 foam micro test tube holders 8
matrix
xylene cyanol DNA loading dye 1 gel staining trays 4
Methodology
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)
19
Your Task:
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.
20
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.
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
plasmid (pGLO), lyophilized 1 inoculation loops, sterile 80
E. coli strain HB101 K-12, 1 micro test tubes, 2.0 ml, sterile, 60
lyophilized color coded
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 1
(CaCl2)
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.
21
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.
22
Laboratory Proper
23
24
13. Analyze results by comparing the appearance of the bacterial samples under UV
light. Tabulate your observations:
EXPERIMENTAL PLATES CONTROL PLATES

+pGLO +pGLO -pGLO -pGLO
LB/amp LB/amp/ara LB/amp LB
3. Maintain the samples by making a stock culture for future use.
Your Task:
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?
25
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.
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
26
Methodology
Use a wavelength of 595 nm.
27
Your Task:
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?
28
3B. Protein Fingerprinting
Materials Needed
pipet tips for gel loading 1 rack Actin and Myosin Standards 1
500 µg
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 30
ml
Laemli sample buffer 30 ml 1
Kaleidoscope prestained 1
standards 500 µl
electrophoresis buffer 1
10x TRIS/glycine/SDS, 1L
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.
29
Your Task:
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?
30
3C. Hydrophobic Interaction Chromatography
Materials Needed

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)
31
Your Task:
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?
32
3D. Enzyme-Linked Immunosorbent Assay (ELISA)
Materials Needed

antigen (chicken ‫צ‬-globulin) 1 microplates with 12-well strips 3
primary antibody (rabit anti- 1 yellow micro test tubes, 2.0 ml 60
chicken polyclonal antibody)
secondary antibody (goat anti- 1 colored micro test tubes, 2 ml 75
body conjugated to horse radish
peroxidase, or HPP)
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
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".
33
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.
34
35
36
Your Task:
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?
37
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.
38
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.
39
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
40
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.
41
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
42
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/
43
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.
44
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
45
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/.
46

Biology 150 Student Lab Manual 2010

Hochgeladen von

Dokumentinformationen

Originalbeschreibung:

Copyright

Verfügbare Formate

Dieses Dokument teilen

Dokument teilen oder einbetten

Freigabeoptionen

Stufen Sie dieses Dokument als nützlich ein?

Sind diese Inhalte unangemessen?

Copyright:

Verfügbare Formate

Biology 150 Student Lab Manual 2010

Hochgeladen von

Copyright:

Verfügbare Formate

Cell and Molecular Biology Laboratory Manual Third Edition

Exercise 1. Basic Instrumentation and Techniques

1. be familiar with the facilities used in cell and molecular biology;

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

Exercise 2. Nucleic Acids

2A. Extraction of Eukaryotic DNA

Each workstation should have the following:

1. Assign one or two representative/s from your group.

Include answers to the following in your report:

2B. DNA Fingerprinting

1. Rehydrate DNA/ buffer samples and restriction enzymes.

2. Rehydrate and aliquot restriction enzymes.

3. Preparation for gel electrophoresis.

5. Maintain a water bath temperature of 45 to 55oC for DNA visualization procedures.

*Caution: Wear protective gloves, goggles and lab gown when

3. Determine the culprit.

4. Document your findings using photodocumentation equipment (camera or

2C. Restriction Fragment Length Polymorphism

2D. Polymerase Chain Reaction

(protocol to be provided by your instructor)

Include answers to the following in your report:

1. What are ALU repeat sequences? What is their evolutionary significance?

3. What are the characteristics of a good PCR primer? Give three.

2E. Organismic Genetics

1. be familiar with some microorganisms used in cell and molecular biology;

a. Fill a 1-L Erlenmeyer flask with 500 ml distilled water.

2. Prepare the arabinose and ampicillin

a. Add 3 ml of transformation solution to the vial containing lyophilized

3. Mark the plates.

a. Label 16 plates "LB".

a. Pour the LB nutrient agar into the 16 plates labeled LB.

6. Rehydrate bacteria and streak starter plates.

a. Obtain lyophilized E. coli strain HB101 K-12.

EXPERIMENTAL PLATES CONTROL PLATES

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

Include answers to the following in your report:

1. What are plasmids? Give two possible advantages and disadvantages of

2. Why is arabinose important in this experiment?

Being biologically all-important, proteins are a much-studied group of diverse molecules.

1. isolate proteins from selected biological sources;

3A. Protein Isolation and Quantitation

Use a wavelength of 595 nm.

Include answers to the following in your report:

3. What is the significance of using a control in this activity?

3B. Protein Fingerprinting

1. Extract fish muscle proteins:

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?

3C. Hydrophobic Interaction Chromatography

Item/s Quantity Item/s Quantity

Prepare Ampicillin and Arabinose Solutions

Prepare Liquid Nutrient Media

1. Put 55 ml distilled water in a 250 ml Erlenmeyer flask and heat to boiling.

(protocol to be provided by your instructor)

Include answers to the following in your report:

1. How can a simple technique like size-exclusion column chromatography exhibit

3D. Enzyme-Linked Immunosorbent Assay (ELISA)

Item/s Quantity Item/s Quantity

Prepare the buffers.