Lab Report 1 Guide
-objective of experiment (one sentence)
-brief summary of experiment (digestion, agarose electrophoresis, and restriction map
-state results of experiment (which plasmid?)
-discuss all concepts for each experiment, technique. (don’t just report what you did in
lab, generally describe these concepts) (restriction enzyme, agarose gel electropherasis)
-must have references
-chemical reactions and mathematical equations (ethidium bromide, log plot of DNA
I highly recommend you read a scientific journal article.
-Write out procedure but make it as short and concise as possible
-Write it in the passive voice (use the zombie test if unsure—see the Lab report
information sheet in the Course information section, general guidelines #6).
-Make sure to include the concentration of all buffers and reagents
-Do not list the reagents you used before the procedure.
-Do not copy and paste it from your protocol.
-Do not include information that a scientist would already know how to do.
-how to make dilutions, set up a reaction, or make buffers.
-how to make a gel or set up a column.
-Do not put data tables in your methods section.
This shows the volume and concentration of each reagent
A single digest was done by adding 5µl of pBluescript plasmid (200ng/µL) to 2µL of Xba I (1U/µL) and
2µL of 10X digestion buffer (1.5 M NaCl, 200mM Tris-HCl pH 7.0) in a total volume of 20uL. A double
digestion was done as above with the addition of 1.5µL of Hind III (3U/µL). All reactions were incubated
at 37°C for 1hr and run on a 1% agarose gel at 80V.
Or, you can be even more concise
This shows the total amount of each reagent by multiplying the volume and the concentration ie. 200ng/uL
x 5uL = 1000ng of total DNA in the reaction
The pBluescript plasmid (1000ng) was digested for 1hr at 37°C with Xba I (2U) alone or with Xba I (2U)
and Hind III (4.5U) in 1X digestion buffer (150mM NaCl, 20mM Tris-HCl pH 7.0). Samples were analyzed
via gel electrophoresis (1% agarose at 80V).
-Include all graphs, gels, calculations, one sample calculation is fine. (gel picture, table to
summarize all bands in the gel and distance measured, log plot, reconstructed plasmid
-Also need to briefly describe results in words. (What does the gel show?).
-Make sure all figures are labeled properly and have titles
-While there are several programs/websites available that will draw a plasmid for you
based upon the information you give it, you MUST DRAW YOUR OWN PLASMID
MAP! This is a learning exercise for you. I am perfectly confident that programs such as
SnapGene can draw a map for you but you will learn very little or nothing by using such
a program. Please use a simple drawing program, make a circle and add hash marks for
your restriction sites.
Fig 1. PCR of Gemin2 Gene
Fig 1a. 1 % Agarose gel of PCR Reactions. The gemin2 gene was amplified from Hela S3 cDNA
with two different sets of primers (one with and with out a stop codon). PCR reactions were run on
gel to determine if the correct products were present. Lane 1 shows the amplification of the Gemin2
gene with a stop codon, Lane2 with out a stop codon, and Lane 3 is a control amplification of the B
actin gene. Fig 1b. shows the DNA mw standard fragments that were run on the gel in a) and the
corresponding base pairs for each fragment.
All three reactions show amplified bands of the correct size, 850bp for the gemin2 gene and 660bp for the
B actin gene. Also in lanes 1 and 2 there was a band that was smaller then 72bp which was due to the
unused primers in the PCR reaction. Other then the unused primers in the reaction there were no other
DNA bands from non-specific PCR amplification in these reactions.
-Interpret your data. (how did you deduce the restriction map from the gel?)
-Were your results as expected and if not, why? (be reasonable)
-Refer back to the specific figures in the results section
The results of the digestion and agarose gel clearly show that there is only one XbaI cut site on the
plasmid. First of all there is only one fragment in the XbaI single digest lane, which is indicative of a
linearized plasmid (Fig 1 lane 3). Furthermore, because the XbaI digested fragment is 4.5kb compared to
the 3.5kb fragment of the undigested super coiled plasmid (Fig1. Lane 1), it shows that the XbaI enzyme did
actually cut the plasmid. The XbaI enzyme cut the super coiled plasmid, which allowed the DNA to
unwind, linearize and consequently the 4.5kb traveled more slowly through gel.
-Limitations of experiments (Please do not include human error).
-Questions from protocol
• Circular, extrachromosomal DNA
Restriction mapping of plasmids
Bacterial “accessory” chromosomes
• Can be passed from one bacteria to
Ability to transfer genes
Importance: antibiotic resistance
• Generally small: ~5-10 kBP
• Origin of replication
• E. coli plasmid must
contain an E. coli origin
• Low = 1-2 copies/cell
• High = 100’s
• Genetic marker
• Restriction sites
• Cleavage of dsDNA at specific base sequence
Degradation of foreign DNA
Host DNA protected by specific methylation
• Restriction sites
Palindromic, 4-8 bp
Sticky ends or blunt ends
• Uses of restriction
Cleavage of large DNA
• Two antibiotic
• DNA fingerprinting
Transfer of genetic
• Hybrid DNA molecules
sites in each gene
Addition of foreign
DNA (cloning) into
• β-galactosidase gene
(lacZ) and ampr
Cloning into lacZ gene:
Use of X-gal in media
• X-gal cleaved by βgalactosidase, then
on a plasmid
Agarose gel electrophoresis
• Separation of nucleic acids
• Anode is + so DNA migrates to it!
1. Digest DNA w/
200-50,000 base pairs
β(1,4) 3,6-anhydro-Lgalactose α(1,3)
100 monomers, ~10kDa
• H-bonding within polygalactose units initiates gel
• Uniform pore size, controlled by agarose
migrates ~500 bp
• MW standards
Gel visualization and measurement
• Ethidium bromide
• Better staining of DNA
• Cost comparison:
stacked bases of dsDNA
Fluoresces under UV
~$0.40/gel ($0.006/gel for EB)
• SYBR Safe gels can go in trash
(so the manufacturer claims)
• EB treated gels must go to
hazardous waste at a cost of
Building a plasmid map
• Restriction enzymes:
Building a plasmid map
Next, use E+H
The 3 fragments are 3 kBP,
1.2 kBP and 0.8 kBP
This means the 3.8 kBP fragment
from H has been cut
Start with the single enzyme that cuts twice (H)
The 2 fragments are 3.8 kBP and 1.2 kBP
Building a plasmid map
Next, use H+S
The 3 fragments are 3.8 kBP,
0.8 kBP and 0.4 kBP
Building a plasmid map
This means the 1.2 kBP fragment
from H has been cut
We still don’t know the relationship
between E & S
This gives us the location of S and
our finished map:
Finally, use E+S
The 2 fragments are 3.4 kBP and
BCH 467: Lab #1
Restriction Map of DNA
Introduction: What color is your DNA?
You will be assigned to investigate an unknown DNA sample: A or B. The DNA contains a
gene encoding either a red fluorescent protein (RFP) or a green fluorescent protein (GFP). In
the first experiment, the DNA will be cut and the resulting fragments separated by agarose
gel electrophoresis. From the sizes of the fragments, you should be able to piece together a
“map” of the DNA. Next week, you will determine the sequence of a portion of the DNA.
By comparing the DNA sequence to the amino acid sequences of the red and green
fluorescent proteins, you should be able to identify the gene. In the third experiment, you
will use a diagnostic PCR test to identify your gene. Combining all of these results should
allow you to give a detailed description of the DNA.
Background information on these types of procedures is covered in Chapters 11 and 12 of the
textbook (Ninfa and Ballou, 1998, Fundamental Laboratory Approaches for Biochemistry
and Biotechnology, pp. 277-299 and pp. 313-323).
A general introduction to fluorescent proteins is given in the article: Matz, M. V., Lukyanov,
K. A., and Lukyanov S. A., 2002, “Family of the green fluorescent protein: journey to the
end of the rainbow”, BioEssays 24, 953-959. More details on the biochemistry of fluorescent
proteins can be found in the review: Zimmer, M., 2002, “Green fluorescent protein (GFP):
applications, structure, and related photophysical behavior” Chem. Rev. 102, 759-781.
Restriction Map of DNA
In the laboratory, DNA is commonly contained in plasmids, which are small, circular pieces of DNA
that can replicate independently of the chromosome. DNA is often manipulated using restriction
enzymes, which cut the DNA at specific recognition sites. The sizes of DNA fragments can be
determined using agarose gel electrophoresis with visualization of the DNA by SYBRsafe staining. In
this experiment, you will use these tools to determine how many sites your plasmid contains for three
different restriction enzymes, and how far apart the sites are on the DNA.
1. L20 pipettor, sterile tips, sterile 1.5 ml eppendorf tubes
eppendorf tube rack, float
2. Plasmid A or B
(50 nanograms/microliter in 1X digestion buffer)
You will investigate either plasmid A or plasmid B. Use the same type of plasmid unknown for
all of the experiments in the DNA section.
3. 1X digestion buffer
(10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 100 mM NaCl, 1 mM 2-mercaptoethanol)
4. Restriction endonucleases BamHI, PstI, and ScaI
(2 units/microliter in 1X digestion buffer, on ice)
(one unit is the enzyme activity that completely cleaves 1 microgram DNA in 1 hour at 37 °C)
Sequences of restriction sites:
5. gel loading solution
(30% glycerol, 10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.025% bromophenol blue)
6. DNA size markers (1 kb ladder, New England Biolabs)
(50 nanograms/microliter in 0.006% xylene cyanol FF, 0.006% bromophenol blue,
0.06% orange G, 2.5% Ficoll 400, 10 mM Tris-HCl, pH 7.9, 10 mM EDTA)
7. 37 °C incubator
8. 0.5X TBE
(44.5 mM Tris base, 44.5 mM boric acid, 1.0 mM EDTA)
9. molten agarose
(1% agarose in 0.5X TBE, boiled and then cooled to ~55 °C)
10. horizontal gel electrophoresis apparatus and power supply
11. staining solution
12. UV imager
1. Set up restriction digests of the plasmid DNA:
Prepare three single digestions and three double digestions with the restriction enzymes BamHI,
PstI, and ScaI, and one sample with no enzyme added. Each digestion should contain 250 ng of
plasmid and 10 units of each enzyme in the digestion in 1X digestion buffer with a total volume of
20 µl. Use sterile eppendorf tubes and sterile pipet tips to avoid contaminating the solutions with
nucleases, and use a fresh tip each time an aliquot is taken from the stocks.
Example of digestion setup:
Plasmid (50 ng/µl)
BamHI (2 U/µl)
PstI (2 U/µl)
ScaI (2 U/µl)
2. Incubate digestion tubes in the 37 °C incubator or heat block for at least 30 minutes.
3. Pour an agarose gel:
Insert the gel casting tray into the casting fixture. Slowly pour 50 ml of molten agarose into the gel
tray (make sure the SYBRsafe has been added by the TA and WEAR GLOVES). Insert the comb
into the slots in the casting tray. Allow the gel to harden undisturbed at room temperature.
4. Set up the gel electrophoresis apparatus:
Add 300 ml 0.5X TBE to the main gel box. When the gel is solidified (fully opaque), transfer the
casting tray to the main gel box. Remove the comb. The running buffer should just cover the gel.
5. Load and run the samples:
When the incubation is done add 5 µl of loading buffer to each sample.
Load 20 µl of each sample in a separate lane on the gel.
Load 10 µl of the size markers in the remaining lane on the gel.
Record the order of the samples in the lanes.
Place the cover on the gel box, connect the electrodes, and set the power supply to 100 volts.
Electrophorese at 100 volts for 1 hour or until the blue dye is approximately 2/3 of the way down the
gel. The size marker contains three dyes: xylene cyanol FF migrates at approximately 4 kb,
bromophenol blue at approximately 300 bp and orange G at approximately 50 bp.
6. Stain and photograph the gel:
When the electrophoresis is finished, bring the gel to the TA, who will photograph the gel using the
Kodak digital gel imaging system.
WEAR GLOVES DURING THIS STEP.
On the photograph of the gel, measure the migration distance from the well to the band for the size
markers and plasmid fragments.
For the size markers, plot log(size in kilobase pairs) versus migration distance or use a semilog plot. Fit
a straight line in the linear region of the size markers, and use this to calculate the sizes of the plasmid
DNA size markers
(1 kb ladder, New England Biolabs)
This image is for your reference only so you
know the sizes of the bands in the ladder. DO
NOT MEASURE THE DISTANCES ON THIS
Using the fragment sizes determined from the analysis of the gel photograph, determine the restriction
map of your plasmid. Show the distances between the restriction sites of the three enzymes.
The plasmid can be thought of as having two parts: the vector and the insert. A vector is a plasmid that
has been engineered to be able to “carry” pieces of DNA. Different genes can be inserted into
restriction sites on the vector. The vectors used for the fluorescent protein genes are called pRSETB and
pQE30. The pRSETB vector contains 1 ScaI site, and the pQE30 vector contains 2 ScaI sites. The
fluorescent protein genes are in the inserts, which do not contain ScaI sites.
Before the next lab period, you should determine whether your vector is pQE30 or pRSETB.
Lab reports must be submitted to SafeAssign through Blackboard. Please include your name
and email address at the top of the first page of your report. Do not include your student ID
number. Any equations and math should be typed using a program such as Mathtype or
Microsoft Equation Editor. All figures should be numbered, clearly labeled and must be
referenced in the text. The file you submit must be a PDF file with a size limit of 2 MB. You
must adhere to the page limits any text beyond the page limit will not be read. LAB REPORTS
MUST BE TYPED IN 12 POINT FONT AND DOUBLE SPACED!
Sections of the Lab Report:
Title (you do not need a title page)
1. Brief project description
2. Most important results and conclusions
1. Objective or purpose of the experiment (what are the goals?)
2. Brief discussion of theory behind the experiments and techniques
3. Any chemical or biochemical reactions
4. Any equations used for analysis of data
Materials and Methods (15%)
1. A narrative of what you did in the lab. In order to write this section, you need to read a
journal article (Journal of Biological Chemistry is a great example).
2. Should include all reagents and instruments used in the experiment.
3. Should enable the reader to repeat your experiment and arrive at similar results
4. Should be clear and concise
5. DO NOT INCLUDE A LIST OR TABLE OF MATERIALS
1. An opening statement to describe the general results obtained
2. Raw data (tables, graphs, photos of gels, etc.) in an organized manner and clearly
labeled figures must be scanned and pasted directly into the text
3. Graphs must be generated with a program such as Excel or Sigma Plot
4. Calculations (refer back to equations mentioned in introduction)
5. Factual description of results
6. Figures and tables must have a title and description
1. Significance of results
2. Discussion of unexpected results, problems encountered, failure of experiment
3. Interpretations supported by data
4. Limitations of data
5. Suggestions for improvement
6. Questions from text
References (may be single spaced)
1. Citations from text
2. Other literature
While there is no grade for this section, lack of references will impact the introduction
Some general guidelines for lab reports:
1. Italics, symbols (w) and sub/superscripts need to be used when appropriate.
2. Don't refer to each week's experiment as a separate week. Think of the lab as one big
study and present it in a logical manner (not necessarily based upon the order in which
you performed the experiments).
3. When using an abbreviation, write out completely one time, then include the abbreviation
in parentheses immediately after. After that, you may use the abbreviation
4. Avoid excessive use of capitalization. Most science
related words (such as polymerase
chain reaction, affinity chromatography, imidazole, etc...) are not capitalized.
5. When reporting standard deviation, it should be done in a manner similar to the
following: 0.567 0.023 mg/ml (note that the units come after the error)
6. The pronouns "T" or "We are not used! The verb tense is always the same. In science,
the passive voice is used, especially in the materials and methods section. If you are
not sure whether or not you are writing in the passive voice, use the zombie test. You
should be able to add the phrase ...by zombies." to the end of any sentence. As an
example of something that passes the zombie test: 10 ul of buffer was added to each of
the reaction tubes (by zombies). This would not pass the zombie test: I added 10ul of
buffer to each reaction tube (by zombies). Use the zombie test and you should never
lose points for failing to use the passive voice.
7. Each sentence should express a single thought and be complete without being a run-on
sentence or having other ghastly grammatical errors.
8. Each paragraph should cover one topic. If you are putting multiple topics into the same
paragraph and that paragraph becomes rather lengthy, you should start a new one.
9. All figures are to be included in the appropriate section. Appendices are not allowed and
will be disregarded by your TA.
10. A good way to write your results and discussion sections is to start by preparing all of
your figures. Then, write these two sections around your figures.
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