General Directives
Questions:
If the answers are not similar to the ones given herein, verify whether it makes sense. There may
be other possible answers which I have not thought about.
Data analysis:

Figures:
These should be properly labeled and easy to understand. They should be large enough
that one can easily interpret them accurately. All figures should be accompanied by a figure
legend on the same or on a different page. These should be numbered and have an appropriate
title. The legend should include sufficient information to know what was done and as required
how. For instance with agarose gels, the markers should be identified, the amount of DNA
loaded, the voltage at which the run was done, the percentage of the gel and any other pertinent
information.
Gels: Restriction digest gels should have ladder, undigested control followed by digests.
Loading should be consistent. Migration should be sufficient for proper analysis. Digest should
show minimal if any partial digests. Overall quality should be good. If the gel from another
group is used, deduct 1 point.
Graphs are figures and should be treated as such. They should be as large as possible for
accuracy. Axes must be labeled and have units. The trend should be hand drawn. Specifically for
standard curves, the Y axis should be a log scale. The numbers should NOT have been changed
to log. The axis should not read “Log of whatever”. As with figures, a figure legend must be
included with a figure number and title. In the case of standard curves it should be stated from
which gel (figure number) the curve was derived from.
 Tables:
These should be numbered and have a title. Ensure that all the information asked for is
given. Verify that the data is in agreement with the figures presented. They should state in their
caption from which figure the data is derived from if there is more than one figure.
1
Assignment 1
Dilutions and Concentrations
Only submit the answers to one digit after the decimal, you do not need to show your
calculations
1. You add water to 125 mL of 1.6 M LiCl to obtain 1.0 L of solution, what is the new
concentration of the solution?
2. In the previous problem, the solution contains how many parts of solute and of solvent?
3. You start with 2.5L of a KNO3 stock solution and wish to prepare 10.0 L of 1.5 M KNO3.
What molarity would the potassium nitrate stock solution need to be if you were to use it all?
4. How many milliliters of 5.0 M copper sulfate solution must be added to 228 mL of water to
achieve a 0.25 M copper sulfate solution?
5. 40.0 mL of 2.0 M Fe(NO3)3 is mixed with 2 mL of 5 M Fe(NO3) and 48 mL of water. What
is the final molar concentration of Fe(NO3)?
6. You add 3.5 L of a HCl solution of unknown concentration to 2.0 L of 0.5 M HCl and obtain
a solution with a final concentration of 1.5 M. What was the unknown concentration of the
initial HCl solution?
7. You have a solution representing 126g/L of NaF (MW: 42g/mole). 180 mL of this solution is
added to water to obtain a final volume of 1080 mL. What is the molarity of the resulting
solution?
8. What is the molar concentration of chloride ions in a solution prepared by mixing 100.0 mL
of 2.0 M KCl with 50.0 mL of a 1.50 M CaCl2 solution?
9. A solution is prepared by dissolving 54 g of AgNO3 in 156 mL of water. What is the percent
concentration (m/m) of AgNO3 of this solution?
10. The A260nm of a DNA solution is 0.12. How much of this DNA solution and a 5.5X loading
dye should you add to 15µL of water to obtain a sample which contains 30ng of DNA in
0.5X loading dye? (A260nm of 1.0 = 50 µg/mL DNA)
1.
2.
3.
4.
5.
0.2M
1 part solute & 7 parts of solvant
6M
12 mL
1M
2
6. 2.1M
7. 0.5M
8. 2.3M
9. 25.7
10. 5µL DNA and 2µL dye
Restriction Mapping
(4 points/question)
1. The linear 12 Kbp DNA fragment shown below has cleavage sites for BamHI and EcoRI.
The numbers indicate the distance in kilobases. Complete the table to indicate the fragment
sizes which would be observed on an agarose gel following each of the indicated digests.
Note, if different fragments of the same size are generated, the size should only be indicated
once. (For example do not indicate 2 and 2Kbp)
B
E
B
E
1
4
6
10
Enzyme digest
Fragment sizes
BamHI
EcoRI
BamHI + EcoRI
2. What fragment sizes could be generated from a BamHI partial digest? Only indicate the
sizes of intermediate fragments which would not be obtained following a complete digest.
3. Draw all possible maps, which could correspond to the results indicated in the above table
for the BamHI digest alone. (DO NOT include mirror images)
4. It was determined that the enzyme XhoI cuts at 0.5Kbp on the map shown above. Indicate
which BamHI fragment would be cut by XhoI and what sizes would be generated in each of
the maps provided for question 3. (Ex. 4Kbp BamHI → 3 + 1 Kbp).
5. A complete digest with EcoRI + BamHI of 12µg of the above fragment was performed.
Indicate the amount in µg of each of the fragments which would be obtained.
3
6. The circular 10Kbp DNA molecule shown below has cleavage sites for BamHI and EcoRI.
Complete the table to indicate the fragment sizes which would be observed on an agarose
gel following each of the indicated digests. Note, if different fragments of the same size are
generated, the size should only be indicated once. (For example do not indicate 2 and 2Kbp)
10/0 Kbp
EcoRI
3 Kbp
BamHI
6 Kbp
BamHI
Enzyme digest
Fragment sizes
BamHI
EcoRI
BamHI + EcoRI
7. What fragment sizes could be generated from a BamHI partial digest? Only indicate the
sizes of intermediate fragments which would not be obtained following a complete digest.
8. Is this the only possible map according to the results presented in the above table? If not
draw another possible map. (Note: Mirror images are not considered different maps)
9. It was determined that the enzyme XhoI cuts at 9Kbp on the map shown above. Indicate
which BamHI fragment would be cut by XhoI and what sizes would be generated in each of
the maps provided for question 8. (Ex. 4Kbp BamHI → 3 + 1 Kbp).
4
10. A 8.9 kb circular plasmid is digested with three restriction enzymes, EcoRI, BamHI and
HindIII, individually and in combination, and the resulting fragment sizes are determined by
means of electrophoresis. The results are as follows:
EcoRI
BamHI
HindIII
EcoRI + BamHI
EcoRI + HindIII
BamHI + HindIII
BamHI + EcoRI + HindIII
8.9 kb
6 kb. 2.9 kb
8.9 kb
6 kb, 2.4 kb, 0.5 kb
7.4 kb, 1.5 kb
5 kb, 2.9 kb, 1 kb
5 kb, 2.4 kb, 1 kb, 0.5 kb
Draw a possible restriction map based on these results. Set the EcoRI site as the origin.
Restriction mapping: (4 points/question)
1.
Enzyme digest
Fragment sizes
BamHI
1, 5, and 6Kbp
EcoRI
2, 4, and 6Kbp
BamHI + EcoRI
1, 2, 3, and 4Kbp
2. 6 and 11 Kbp
3. (1,5,6), (6,1,5), and (5,6,1), note reverse orders are accepted. Only three possibilities.
4.
1 → 0.5
5 → 0.5 + 4.5
6 → 0.5 + 5.5
5. 1 µg of 1Kbp
4 µg of 2Kbp
3 µg of 3Kbp
4 µg of 4Kbp
6.
Enzyme digest
Fragment sizes
BamHI
3 and 7Kbp
EcoRI
10Kbp
BamHI + EcoRI
3 and 4Kbp
5
7. 10Kbp
8. Only this map is possible.
9. 3 → 1 + 2
7→6+1
10. Any rotation and mirror image of this map are valid.
8.9/0 Kbp
EcoRI
8.4 Kbp BamHI
BamHI 2.4 Kbp
7.4 Kbp HindIII
6
Lab exercise
(4 points/question)
Dilution Exercise:
1.
A 1.5mM solution of compound “A”. 0.12ml Soln. 1 + 0.88ml water
A 0.36% (m/v) solution of compound “B”. 0.05ml soln. 2 + 0.95ml water
A 6% (v/v) solution of solution I. 0.06ml soln 1 + 0.94ml water
A solution containing 0.5mg of compound “A” and 0.1% (v/v) of compound “B”.
0.05ml soln. 1, 0.022ml soln. 2 + 0.93ml water
e. A solution representing the following ratio: solution I: solution II : water : 2:1:2
0.4ml soln. 1, 0.2ml soln. 2 + 0.4ml water
2. Indicate the average absorbencies of each of the 4 solutions you prepared. (1 point )
Should be within 25% of the following values to obtain points:
a.
b.
c.
d.
0.551
0.186
0.291
0.324
a.
b.
c.
d.
Determining DNA concentration:
1. Submit a table of the DNA concentration determinations experiment performed on page 13
of the lab manual. Your table should include the following information: Standard DNA
concentrations (µg/mL), corresponding A260 readings, and A260 readings of each of the
unknown diluted solutions of DNA you prepared. Check general directives. Verify that
absorbance readings are consistent with the dilution.
2. Submit a graph representing DNA concentration Vs A260 readings. Include a line of best fit,
The R2 coefficient, and the formula of the line. Check general directives. R2 coefficient
should be greater than 0.95
3. According to the graph, what is the constant which represents the relationship between an
A260 of 1.0 and DNA concentration in µg/mL. (Ex. A260 of 1.0 = X µg/mL) Should be
50µg/mL +/- 20%
4. According to the constant determined in the previous question, what was the DNA
concentration of the original unknown DNA solution provided? Should be consistent with
value given in Q. 3, approx 0.06 µg/ul
Agarose gel electrophoresis and restriction digests:
1. Submit a figure and an appropriate figure legend of the agarose gel described on page 16 of
the lab manual. Check general directives.
2. Submit a standard curve of the Molecular weight ladder (Size in Kbp Vs. migration
distance) Check general directives.
3. Submit a table of the restriction digests of the recombinant plasmid which includes the
following information: Enzyme used, number of cuts, fragment sizes observed. In a caption
accompanying the table submitted, indicate the total size of the plasmid, the size of the
vector, the size of the insert, and the restriction site (s) in which the insert was introduced in
the vector. Check general directives.
7
4. Provide a figure which represents a possible restriction map of the insert within the multiple
cloning site of pUC9. Your map should be linear and only include the insert within the
multiple cloning site. Check general directives.
8
Assignment 2
Restriction digests and mapping (4 points/question)
1. Define the following terms: Isoschizomer, neoschizomer, and isocaudomer.
Isoschizomers: restriction enzymes specific to the same recognition sequence and cut in the
same location.
Neoschizomer: An enzyme that recognizes the same sequence but cuts it at a different position.
Isocaudomer: An enzyme that recognizes a different sequence, but produces the same ends.
The table below presents the results of different digests of a plasmid. Note, that according to the
gel from which the data was derived, the BamHI digest may be partial.
HindIII
BamHI
EcoRI
HindIII + BamHI
HindIII + EcoRI
BamHI + EcoRI
3.82, 0.18
4.0, 2.35, 1.65
3.0, 1.0
3.55, 2.35, 1.2, 0.27, 0.18
1.87, 1.0, 0.95, 0.18
1.6, 1.4, 1.0, 0.75, 0.25
2. What is the total plasmid size? 4Kbp
3. Are the digests with BamHI partial or complete? Justify your answer.
Partial; the size adds up to more than the total size of 4Kbp.
4. Which fragments in the BamHI, if any, represent products from a partial digest? 4Kbp
5. Draw a circular map which is in agreement with the results presented. (8 points)
HindIII
6. Given that 1µg of DNA was used for the EcoRI digest, what are the approximate quantities in
µg of each of the fragments? 3.0Kbp: 0.75µg and 1.0Kbp :0.25µg
9
7. The following list represents a list of commonly used restriction enzymes:
a. SalI (G/TCGAC)
b. EcoRI (G/AATTC)
c. BclI (T/GATCA)
d. BamHI (G/GATCC)
e. XhoI (C/TCGAG)
f. PstI (CTGCA/G)
g. ClaI (AT/CGAT)
h. NarI (GG/CGCC)
Indicate which enzyme pairs generate compatible cohesive ends.
SalI + XhoI; BclI + BamHI; ClaI + NarI
8. The restriction Enzyme ApoI cleaves the sequence R/AATTY (R= A or G and Y = C or T).
How many different palindromes does ApoI recognize and cleave? 2
9. A DNA fragment generated with the restriction enzyme XbaI (T/CTAGA) was inserted into
the unique NheI (G/CTAGC) site of a vector. Could the new recombinant plasmid be
digested with XbaI, NheI, or both enzymes to release the insert? Justify your answer.
Neither, the new site does not correspond to either restriction site: GCTAGA
You performed restriction digests and agarose gel electrophoresis of a plasmid using 3 different
restriction enzymes. The gel is shown below. Unfortunately, you forgot to label your tubes. The
only things you remember is that your standards are in Lane 5 and your uncut control is in Lane
1. Also, you loaded the same amount of total DNA in all the sample wells (1-4).
1
2
3
4
5
16Kb
8 Kb
2.5 Kb
2.0 Kb
2
1.0 Kb
0.5 Kb
10. What is the approximate size of the plasmid?
 20 kb
 16 kb
 6.5 kb
 5.0 kb
11. How many times did the enzyme used in Lane 2 digest the plasmid? Does the data seem
reasonable? Justify. 2X, yes its a double band
10
12. How many times did the enzyme used in Lane 4 digest the plasmid? Does the data seem
reasonable? Justify. 3X; 2Kbp band is a partial and 1 Kbp band is 2 fragments.
Lab Exercises
(4 points/question)
Gel electrophoresis of plasmid purifications and restriction digests of pBR322 (pg 24):
1. Submit a figure representing your agarose gel. Make sure to include an appropriate legend.
Follow the directives for figures on the web page of this course. Make sure to include all the
required information in the legend for the understanding and interpretation of the figure.
Check general directives.
2. Based on the results obtained from your restriction digests, answer the following questions:
a. How many times did PvuII cut within the plasmid? (once)
b. How many times did HincII cut within the plasmid? (Twice)
c. How many times did HincII cut within the PvuII fragment? (Twice)
d. What are the distances between the PvuII and the HincII sites? approx. 1415 & 1840
11
Project I: Verifying the restriction map of a DNA insert
1. Submit a figure representing the agarose gel electrophoresis of your single digests. Make
sure to include an appropriate legend. Follow the directives for figures on the web page of
this course. Make sure to include all the required information in the legend for the
understanding and interpretation of the figure. Check general directives.Should be similar to
this depending on the orientation they had:
1.
2.
3.
4.
5.
6.
7.
8.
12
Mol wt. marker
Uncut
BamH1
HindIII
PstI
PvuII
ScaI
pUC Bam
2. Submit a figure representing the agarose gel electrophoresis of your double digests. Make
sure to include an appropriate legend. Follow the directives for figures on the web page of
this course. Make sure to include all the required information in the legend for the
understanding and interpretation of the figure. Check general directives.
3. Submit a table presenting the analysis of the restriction digests. Your table should include:
Enzyme (s) used, Total number of cuts, Number of cuts in the vector, Number of cuts in the
insert, and Fragments sizes generated. Check general directives.
4. Submit a figure of the restriction map of the insert. Your map must be linear, include the
multiple cloning site, indicate the insertion site, the size of the insert, the positions in the
multiple cloning site or the insert of all the enzymes tested. Your figure must be to scale.
Follow the directives for generating such a figure under the heading Graphs/Figures on this
course's web site. Check general directives.
5. The enzyme XhoI cuts once within the unknown DNA insert you have been working with.
Given this information and the map you have generated, design a proper experiment which
would allow you to determine the precise position of the XhoI site. Your experiment must
include the following information:
a. What samples would be loaded on the gel?
b. What single and or double digests would be performed?
c. What fragment sizes would be predicted, if known?
d. How would these digests allow you to determine the position of the XhoI
restriction site?
Should include undigested, single digests for each enzyme and double digests. HindIII is best
choice for second enzyme.
13
Bioinformatics 1: (4 points)
1. Submit a table with the following information with regards to each of the unknown genes
available under the heading Sequences/UNKNOWN GENES on this course’s web site.








The accession number (#2)
Coverage
Max Ident.
E value
The definition (#1)
The organism from which this sequence was obtained (#3)
The product of the gene (#4)
The protein id. This is the protein’s accession number (#5)
Accession
Y00417
Organism
Triticum aestivum
X01108
Triticum aestivum
AB022060
Triticum aestivum
X02352
Triticum aestivum
X15944
Triticum aestivum
Accession
Y00417
X01108
AB022060
X02352
X15944
Coverage
42%
25%
18%
31%
30%
Definition
Wheat mitochondrial COI gene
for cytochrome oxidase subunit I
Wheat mitochondrial cytochrome
oxidase subunit II (CO II) gene
Triticum aestivum mitochondrial
gene for ORF25
Wheat mitochondrial gene for
apocytochrome b
Triticum aestivum mitochondrial
cox3 gene
Max ident.
100%
100%
99%
99%
100%
E value
0
0
0
0
0
14
Protein id.
CAA68474.1
BAA82046.1
Gene product
cytochrome oxidase
subunit I
cytochrome oxidase
subunit II
ORF25
CAA26207.1
apocytochrome b
CAA34071.1
cytochrome oxidase
subunit III
CAA25581.1
Bioinformatics 2
Restriction mapping (4 points/question)
1. Present theoretical maps of all unknown genes. Indicate below each map the name of the
gene and list the enzymes which do not cut.
2. Compare the theoretical maps generated above to the experimental map of the unknown
insert you analyzed in Project 1. The unknown insert corresponds most closely to which
gene? Cox2
3. Submit a printout of the FASTA sequence of the gene corresponding to the unknown insert.
4. Submit a table indicating how many times each of the following enzymes cut within the
unknown insert: AccI, BglII, MboI, NcoI, and NotI.
5. Amongst the enzymes indicated in the previous question, which one cuts the most often
within the DNA insert? Give a reason which would explain why this enzyme cuts more often
than the others. MboI, recognition sequence is only 4 bases therefore more probable than
others which are 6 - 8 bases.
15