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Assignment 1
Part I: Dilutions and Concentrations (3 points/ question for a total of 60 points)
1 0.083M
2 25 mL
3 0.025%
4 5%
5 24 parts
6 6.28%
7 24:5:2:1
8 23.3:5.1:2.14:1
9 60.6%
10 50 mL
11 3 M
12 4 M
13 907.4 mL
14 2.33M
15 10 µL DNA and 3 µL Dye
16 1881.13 µg/mL
17 5.36X
18 646 µg/L
19 3.9g K and 4.75g PO4
20 158.65 mL
Part II: Lab performance and data analysis (5 points/question for a total of 40 points)
1. Indicate the absorbance readings obtained for each of the following solutions.
Solution ABS
a. A 0.2 mM solution of compound “A”. 0.72
b. A 0.72% (m/v) solution of compound “B”. 0.67
c. A 5% (v/v) solution of solution I. 0.341
d. A solution containing 0.1 mg of compound “A” and 0.1% (v/v) of compound “B”. 1.5
e. A solution with the following ratio: solution I: solution II : water : 2 : 1 : 247 1.3
1 point each for values within 10% of those indicated
0.5 point for values within 30% of those indicated
0 points for values beyond 30% of those indicated
1.3: Using micropipettors
2. Generate a standard curve from the data obtained with the volumes ranging from 50-200 µL.
(ABS Vs Vol.). Determine the R2 coefficient. (Follow the directives for figures and graphs
available on this course’s web site)
Criteria for grading graphs:
Has a title
Axis labels with units
Trend line with equation and R2 coefficient are indicated
R2 coefficient is 0.95 or greater
3. According to your standard curve, what were the average volumes for wells G1-3 or H1-3 and
wells G4-6 or H4-6? Give points if done appropriately according to data obtained
1.4: Colorimetric assay to determine phosphate concentration
4. Generate a standard curve. (ABS Vs µmoles phosphate per well). Determine the R2 coefficient.
(Follow the directives for figures and graphs available on this course’s web site)
Criteria for grading graphs:
Has a title
Axis labels with units
Curve and R2 coefficient for linear portion are indicated
R2 coefficient is 0.95 or greater
2.3: Agarose gel electrophoresis
5. Submit an appropriate figure of your gel electrophoresis including an accompanying legend.
(Follow the directives for figures and graphs available on this course’s web site)
Criteria for grading gels:
Has a figure title
Lanes are labelled
Sizes of molecular weight standards are indicated
Legend with required information for interpretation of gel
o Legends should provide enough information so that the figure is understandable
and the experiment can be reproduced by someone who is familiar with molecular
biology. Define all symbols used in the figure and define all abbreviations.
Quality of migration: consistent loading, clear gel, migration distance is adequate
For plasmid isolations obtained a decent yield, with little to no RNA
2.4: Spectrophotometric quantification of DNA
6. Submit a graph representing the A260 readings Vs standard DNA concentrations. Add a
trend line of best fit. Determine the equation of the line and the R2 coefficient. (Follow the
directives for figures and graphs available on this course’s web site)
2.5 points
2.5 points
2.5 points
2.5 points
2.5
poin
ts
2.5 points
Criteria for grading graphs:
Has a title
Axis labels with units
Trend line with equation and R2 coefficient are indicated
R2 coefficient is 0.95 or greater
7. According to your graph what was the DNA concentration of the undiluted unknown DNA
solution provided? Give points if done appropriately according to data obtained
8. Provide the following information for your yeast genomic DNA isolation:
ABS260
Concentration in µg/µL of undiluted preparation
o 2.5 points for conc. between 150 - 400 µg/mL
o 1 point for higher or lower conc.
Total yield in µg (2.5 points if done correctly)
2.5 points
2.5 points
Assignment #2
Part I: Restriction digests and mapping (2 points/ question for a total of 50 points)
1. Bacillus
2.
Isoschizomer: Pairs of restriction enzymes specific to the same recognition sequence and
which generate the same termini.
Neoschizomer: Pairs restriction enzymes that recognize the same nucleotide sequence but
cleave at a different site generating different termini.
Isocaudomer: Pairs of restriction enzymes with different recognition sequences but
generate identical termini.
3.
A+D
A+B
B+D
C+F
4. Any of the following
CT/AATTAG A/AATTT
CTA/ATTAG AA/ATTT
CTAATT/AG AAATT/T
CTAAT/TAG AAAT/TT
5. Neither A or B
6.
a. 1
b. 2
c. 2
d. 7
7. BamHI 1, 5 and 12
EcoRI 2, 4 and 6
BamHI + EcoRI 1, 2, 3, 4
Enzyme Recognition Sequence
A AccI GT/CGAC
B ClaI AT/CGAT
C EagI C/GGCCG
D TaqI T/CGA
E NsiI ATGCA/T
F NotI GC/GGCCGC
G PstI 5CTGC/AG
8. 6 and 11
9. 5 kb
10. 4 µg
11. BamHI
12. 13199 or 12126 or 9645 or 10701
13. 13199
14. 1425 + 11774
15. 2498 + 2129
16. 1073 + 3554
17. 3242
18. 1855 + 1387
19. 2934
20. 2934
21. 2
22. 2048
23. True
24. 29 times
25. TaqI
Part II: Restriction mapping (4 points/ question for a total of 40 points) 1. Submit a standard curve of the molecular weight ladder (Migration distance Vs. Size in Kbp)
1 points/criteria listed below
Figure number & Title provided (Title should be in caption)
Appropriate axis labels with units are provided
Y axis represents log of base pairs
Trend line provided (may be drawn by hand) which has a good linear range.
Either line of best fit or curve is acceptable.
2. Submit a table presenting the results of the restriction digests of the recombinant plasmid
Note sizes are approximate. (2 points) Accept 20% range as long as data is in agreement
between digests.
Enzyme Total cuts Cuts in insert Cuts in vector Sizes in Kb
BamHI 2 1 1 4.0, 0.7
EcoRI 2 1 1 3.5, 1.2
HindIII 2 0 2 2.9, 1.8
PstI 1 0 1 4.7
EcoRI + HindIII 3 1 2 2.9, 1.0, 0.8
Information provided in caption (2 points) Accept 20% range as long as data is in agreement with
data in table.
Total size in the range of 4.7 Kb
Size of vector in the range of 2.9 Kb
Size of insert in the range of 1.8Kb
Insertion site: HindIII
3. Submit a figure which represents a possible restriction map of the insert within the multiple
cloning site of pUC9.
Figure provided with Accompanying legend (caption)
Figure number and Figure title provided (caption)
Computer drawn or hand drawn linear map
Should be to scale
Must include scale bar
Should indicate following regions: multiple cloning site and insert
Insertion site should be easily determined from figure
Map is in agreement with data presented in table
Example:
4. Submit a table presenting the analysis of the restriction digests of the unknown you were
provided with. 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.
Table provided with information requested.
Title and table number provided
5. Submit a figure of the restriction map of the insert from the recombinant plasmid you were
provided with. Your map must be linear, include the multiple cloning site, 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. (Make sure that the enzyme indicated as the insertion
site is consistent with both orientations) Follow the directives for generating such a figure
under the heading Graphs/Figures on this course's web site.
Figure provided with Accompanying legend (caption)
Figure number & Figure title provided (caption)
Computer drawn or hand drawn linear map
Should be to scale
Must include scale bar
Should indicate following regions: multiple cloning site and insert
Insertion site should be easily determined from figure
Map is in agreement with data presented in table
6. Submit a figure of your own agarose gel electrophoresis of the predigested pUC recombinant
and the calibration of a restriction enzyme. Make sure to include an appropriate legend. Follow
the directives for figures on the Web page of this course and to include all the required
information in the legend for the understanding and interpretation of the figure.
Criteria for grading gel:
Figure provided with Accompanying legend
Figure number & Figure title provided
Lanes are labelled and easy to understand
First lane is mw ladder
Second lane is undigested control
MW sizes of standard are indicated
Gel clearly shows bands and migration is satisfactory
Legend indicates parameters of migration: Voltage, agarose concentration, samples
loaded
o Legends should provide enough information so that the figure is understandable
and the experiment can be reproduced by someone who is familiar with molecular
biology. Define all symbols used in the figure and define all abbreviations.
Quality of migration: consistent loading, clear gel, migration distance is adequate
7. According to the experiment presented in question 6, what was the most dilute sample which
showed a complete digestion (Indicate the dilution)? Based on this information, what was the
approximate undiluted enzyme concentration in units/µL? Show how you arrived to this
conclusion.
Digestion occurred
Correct dilution was chosen to determine concentration
Units/µL should be equal to reciprocal of dilution chosen X 0.5/2
2 p
oin
ts
2 points
Submit a figure of your own agarose gel electrophoresis of the restriction digests of the
recombinant pUC plasmid you were provided with.
Criteria for grading gel:
Figure provided with Accompanying legend
Figure title provided
Lanes are labelled and easy to understand
First lane is mw ladder
Second lane is undigested control
MW sizes of standard are indicated
Gel clearly shows bands and migration is satisfactory
Legend indicates parameters of migration: Voltage, agarose concentration, samples
loaded
o Legends should provide enough information so that the figure is understandable
and the experiment can be reproduced by someone who is familiar with molecular
biology. Define all symbols used in the figure and define all abbreviations.
Quality of migration: consistent loading, clear gel, migration distance is adequate
8. BamHI (1), EcoRI (0)
9. Draw a possible restriction map of this genomic region of S. cerevisiae.
Computer drawn or hand drawn linear map
Should be to scale
Must include scale bar
Sizes are very approximate, anything relatively close is acceptable. Order is most important
Either orientation is acceptable
Bioinformatics 1-2 (1.25 points/ question for a total of 10 points) 1. AEB64941 (Decimal is unimportant)
2. Nucleotide sequence
3. polA
2 p
oin
ts
2 points
Bam Bam Bam Eco Eco
1200 650 850 appox 25 000
4. Submit the following information with regards to each of the unknown genes from the first
bioinformatics exercise.
Acc. Num. Cov. Ident. E value Definition Organism Gene name Product Pro. Acc.
NM_057444.3 100% 100% 0.0 Drosophila
melanogaster yellow
(y), mRNA
Drosophila
melanogaster
y yellow NP_476792.1
X91249.1 100% 100% 0.0 H.sapiens mRNA for
white gene protein
Homo sapiens White Non provided CAA62631.1
XM_019107967.1 99% 93% 0.0 Cyprinus carpio
mitogen-activated
protein kinase 8B-like
Cyprinus
carpio
LOC109094263 mitogen-activated
protein kinase 8B-
like
XP_018963512.1
Y00417 42% 100% 0.0 mitochondrial
cytochrome C oxidase
subunit I
Triticum
aestivum
Non indicated or
COI
cytochrome oxidase
subunit I
CAA68474.1
XM_006340335.2 99% 92% 0.0 Solanum tuberosum
uncharacterized
LOC102604569
Solanum
tuberosum
LOC102604569 uncharacterized
protein
LOC102604569
XP_006340397.1
5. Provide theoretical restriction maps of the 5 unknown genes available on this course’s Web
page. Indicate below each map the name of the gene and list the enzymes that do not cut.
Give points if done and includes required info.
6. COI or cytochrome oxidase 1
7. NcoI does not cut.
8. 645, 648, 269, 235 and 106
Assignment #2
Part I: Restriction digests and mapping (2 points/ question for a total of 50 points)
26. Bacillus
27.
Isoschizomer: Pairs of restriction enzymes specific to the same recognition sequence and
which generate the same termini.
Neoschizomer: Pairs restriction enzymes that recognize the same nucleotide sequence but
cleave at a different site generating different termini.
Isocaudomer: Pairs of restriction enzymes with different recognition sequences but
generate identical termini.
28.
A+D
A+B
B+D
C+F
29. Any of the following
CT/AATTAG A/AATTT
CTA/ATTAG AA/ATTT
CTAATT/AG AAATT/T
CTAAT/TAG AAAT/TT
30. Neither A or B
31.
e. 1
f. 2
g. 2
h. 7
32. BamHI 1, 5 and 12
EcoRI 2, 4 and 6
BamHI + EcoRI 1, 2, 3, 4
Enzyme Recognition Sequence
A AccI GT/CGAC
B ClaI AT/CGAT
C EagI C/GGCCG
D TaqI T/CGA
E NsiI ATGCA/T
F NotI GC/GGCCGC
G PstI 5CTGC/AG
33. 6 and 11
34. 5 kb
35. 4 µg
36. BamHI
37. 13199 or 12126 or 9645 or 10701
38. 13199
39. 1425 + 11774
40. 2498 + 2129
41. 1073 + 3554
42. 3242
43. 1855 + 1387
44. 2934
45. 2934
46. 2
47. 2048
48. True
49. 29 times
50. TaqI
Part II: Restriction mapping (4 points/ question for a total of 40 points) 10. Submit a standard curve of the molecular weight ladder (Migration distance Vs. Size in Kbp)
1 points/criteria listed below
Figure number & Title provided (Title should be in caption)
Appropriate axis labels with units are provided
Y axis represents log of base pairs
Trend line provided (may be drawn by hand) which has a good linear range.
Either line of best fit or curve is acceptable.
11. Submit a table presenting the results of the restriction digests of the recombinant plasmid
Note sizes are approximate. (2 points) Accept 20% range as long as data is in agreement
between digests.
Enzyme Total cuts Cuts in insert Cuts in vector Sizes in Kb
BamHI 2 1 1 4.0, 0.7
EcoRI 2 1 1 3.5, 1.2
HindIII 2 0 2 2.9, 1.8
PstI 1 0 1 4.7
EcoRI + HindIII 3 1 2 2.9, 1.0, 0.8
Information provided in caption (2 points) Accept 20% range as long as data is in agreement with
data in table.
Total size in the range of 4.7 Kb
Size of vector in the range of 2.9 Kb
Size of insert in the range of 1.8Kb
Insertion site: HindIII
12. Submit a figure which represents a possible restriction map of the insert within the multiple
cloning site of pUC9.
Figure provided with Accompanying legend (caption)
Figure number and Figure title provided (caption)
Computer drawn or hand drawn linear map
Should be to scale
Must include scale bar
Should indicate following regions: multiple cloning site and insert
Insertion site should be easily determined from figure
Map is in agreement with data presented in table
Example:
13. Submit a table presenting the analysis of the restriction digests of the unknown you were
provided with. 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.
Table provided with information requested.
Title and table number provided
14. Submit a figure of the restriction map of the insert from the recombinant plasmid you were
provided with. Your map must be linear, include the multiple cloning site, 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. (Make sure that the enzyme indicated as the insertion
site is consistent with both orientations) Follow the directives for generating such a figure
under the heading Graphs/Figures on this course's web site.
Figure provided with Accompanying legend (caption)
Figure number & Figure title provided (caption)
Computer drawn or hand drawn linear map
Should be to scale
Must include scale bar
Should indicate following regions: multiple cloning site and insert
Insertion site should be easily determined from figure
Map is in agreement with data presented in table
15. Submit a figure of your own agarose gel electrophoresis of the predigested pUC recombinant
and the calibration of a restriction enzyme. Make sure to include an appropriate legend. Follow
the directives for figures on the Web page of this course and to include all the required
information in the legend for the understanding and interpretation of the figure.
Criteria for grading gel:
Figure provided with Accompanying legend
Figure number & Figure title provided
Lanes are labelled and easy to understand
First lane is mw ladder
Second lane is undigested control
MW sizes of standard are indicated
Gel clearly shows bands and migration is satisfactory
Legend indicates parameters of migration: Voltage, agarose concentration, samples
loaded
o Legends should provide enough information so that the figure is understandable
and the experiment can be reproduced by someone who is familiar with molecular
biology. Define all symbols used in the figure and define all abbreviations.
Quality of migration: consistent loading, clear gel, migration distance is adequate
16. According to the experiment presented in question 6, what was the most dilute sample which
showed a complete digestion (Indicate the dilution)? Based on this information, what was the
approximate undiluted enzyme concentration in units/µL? Show how you arrived to this
conclusion.
Digestion occurred
Correct dilution was chosen to determine concentration
Units/µL should be equal to reciprocal of dilution chosen X 0.5/2
2 p
oin
ts
2 points
Submit a figure of your own agarose gel electrophoresis of the restriction digests of the
recombinant pUC plasmid you were provided with.
Criteria for grading gel:
Figure provided with Accompanying legend
Figure title provided
Lanes are labelled and easy to understand
First lane is mw ladder
Second lane is undigested control
MW sizes of standard are indicated
Gel clearly shows bands and migration is satisfactory
Legend indicates parameters of migration: Voltage, agarose concentration, samples
loaded
o Legends should provide enough information so that the figure is understandable
and the experiment can be reproduced by someone who is familiar with molecular
biology. Define all symbols used in the figure and define all abbreviations.
Quality of migration: consistent loading, clear gel, migration distance is adequate
17. BamHI (1), EcoRI (0)
18. Draw a possible restriction map of this genomic region of S. cerevisiae.
Computer drawn or hand drawn linear map
Should be to scale
Must include scale bar
Sizes are very approximate, anything relatively close is acceptable. Order is most important
Either orientation is acceptable
Bioinformatics 1-2 (1.25 points/ question for a total of 10 points) 5. AEB64941 (Decimal is unimportant)
6. Nucleotide sequence
7. polA
2 p
oin
ts
2 points
Bam Bam Bam Eco Eco
1200 650 850 appox 25 000
8. Submit the following information with regards to each of the unknown genes from the first
bioinformatics exercise.
Acc. Num. Cov. Ident. E value Definition Organism Gene name Product Pro. Acc.
NM_057444.3 100% 100% 0.0 Drosophila
melanogaster yellow
(y), mRNA
Drosophila
melanogaster
y yellow NP_476792.1
X91249.1 100% 100% 0.0 H.sapiens mRNA for
white gene protein
Homo sapiens White Non provided CAA62631.1
XM_019107967.1 99% 93% 0.0 Cyprinus carpio
mitogen-activated
protein kinase 8B-like
Cyprinus
carpio
LOC109094263 mitogen-activated
protein kinase 8B-
like
XP_018963512.1
Y00417 42% 100% 0.0 mitochondrial
cytochrome C oxidase
subunit I
Triticum
aestivum
Non indicated or
COI
cytochrome oxidase
subunit I
CAA68474.1
XM_006340335.2 99% 92% 0.0 Solanum tuberosum
uncharacterized
LOC102604569
Solanum
tuberosum
LOC102604569 uncharacterized
protein
LOC102604569
XP_006340397.1
9. Provide theoretical restriction maps of the 5 unknown genes available on this course’s Web
page. Indicate below each map the name of the gene and list the enzymes that do not cut.
Give points if done and includes required info.
10. COI or cytochrome oxidase 1
11. NcoI does not cut.
12. 645, 648, 269, 235 and 106
Assignment #3
Part I: Cloning and transformations (2.5 points/ question for a total of 50 points) 1.
Heterozygous carrier mother 7600 + 13000
Homozygous normal mother 7600
Homozygous for sickle cell anemia fetus 13000
Combined genomic DNA from a heterozygous carrier mother + homozygous normal father
7600 + 13000
2. Set 3
3. Directional cloning: StuI + SalI
4. Non directional cloning: SalI
5. EcoRI
6. 2
7. 3
8. b
9. 1.95 X 103
10. NdeI
11. NcoI
12. 7 kb
13. 1 + 6kb in either orientation
14. 0.4, 1.1, 1.5, 2.7 and 4.1kb
15. 2.3, 2.7, 3.0, 4.1 and 5.3 kb
16. 1.5, and 5.3
17. 3.4 kb
18. C + E
19. 9 times
20. A
Part II: Cloning (5 points/ question for a total of 25 points)
1. Submit a figure representing your PCR of the GFP gene. Include an appropriate legend which
includes the size of the amplicon observed.
Criteria for grading gel:
Figure provided with Accompanying legend
Figure title provided
MW sizes of standard are indicated
Legends should provide enough information so that the figure is understandable and the
experiment can be reproduced by someone who is familiar with molecular biology. Size
of amplicon is indicated
Obtained an amplification product of adequate yield
2 p
oin
ts
3 points
2. Give points if done
Total number of colonies observed
Number of white colonies
Number of blue colonies
Number of green fluorescent colonies
3. Give points if calculation was done correctly
4. Submit a figure of your PCR analysis of your plasmid recombinants.
Criteria for grading gel:
Figure provided with Accompanying legend
Figure title provided
MW sizes of standard are indicated
Legends should provide enough information so that the figure is understandable and the
experiment can be reproduced by someone who is familiar with molecular biology. Size
of amplicon is indicated. Briefly explains if the sizes obtained are those expected and
whether the results indicate that the amplicon was cloned in the correct orientation.
Obtained amplification products
5. Submit a figure of your restriction enzyme agarose gel electrophoresis of your plasmid
recombinant.
Criteria for grading gel:
Figure provided with Accompanying legend
Figure title provided
MW sizes of standard are indicated
Lanes are labelled and easy to understand
First lane is mw ladder
Second lane is undigested control
Legends should provide enough information so that the figure is understandable and the
experiment can be reproduced by someone who is familiar with molecular biology. Size
of amplicon is indicated. Briefly explains if the sizes obtained are those expected and
whether the results indicate that the amplicon was cloned in the correct orientation.
Loading is consistent between lanes
Gel clearly shows bands and migration is satisfactory
Digestions were adequate
3 p
oin
ts
2 points
2 p
oin
ts
2 points
Bioinformatics 3 (2.5 points/ question for a total of 25 points)
1. 5’ TAATCACACCTTTTTGTTTC 3’
2. 3’ GATAAGATGGGATCCGATC 5’
3. The following primers were used in exercise 4 to amplify and mutagenize the GFP gene using
pGFPuv as a template (the pGFPuv sequence can be obtained on this course’s Web site):
+ 230 CGCCAAGCTT-GCATGCCTGCAGGTCG 255 GFP
|||||||||| ||||||||||||||||
+ 1 CGCCAAGCTTTGCATGCCTGCAGGTCG 27 GFPfor-1
+ 230 CGCCAAGCTTGCATGCCTGCAGGTCG 255 GFP
||||||||||||||||||||||||||
+ 1 CGCCAAGCTTGCATGCCTGCAGGTCG 26 GFPfor-2
+ 230 CGCCAAGCTTG--CATGCCTGCAGGTCG 255 GFP
|||||||||| |||||||||||||||
+ 1 CGCCAAGCTTTGACATGCCTGCAGGTCG 28 GFPfor-3
+ 1130 CTGACACATGCAGCTCCCGGAGACGG 1155 GFP
||||||||||||||||||||||||||
- 26 CTGACACATGCAGCTCCCGGAGACGG 1 GFPrev
4. 925 base pairs
5. GFPSc-F1 + GFPSc-R1
6. 915 bp
7. No amplicon expected
8. Map the alignment positions of each of the following primer sequences on the sequence of the
pUC19 sequence. You may obtain the pUC19 sequence on the course’s Web site.
9. B + D
10. Sequence of forward primer and restriction site added. (SphI) GCATGC ATGGCCCTG
Sequence of reverse primer and restriction site added. (EcoRI) GAATTC CTAGTTGCA
Expected size of amplicon. 345 bp
pUC19
147 2425 2485
152
Bioinformatics 4 & 5 (1 point/ question for a total of 25 points) 1. NM_001077422.3
2. NM_001004376.3
3. NM_000559.2
4. What is the percentage of identity at the nucleotide level between the hemoglobin genes from
each of the following pairs of organisms:
A. 82.39%
B. 65.52%
C. 67%
D. 53.73%
5.
Human alpha hemoglobin to chicken hemoglobin
Cow hemoglobin to chicken hemoglobin
Human alpha hemoglobin to human fetal hemoglobin
6. None
7. What is the percentage of identity at the protein level between the hemoglobin proteins from
each of the following pairs of organisms:
A. 88.03%
B. 70.42%
C. 71.13%
D. 33.10%
8. XP_010804269.1
9. NP_033756.2
10. AAA34411.1
11.
Bos Taurus to yeast
Human to yeast
Mouse to yeast
12.
Bos Taurus to human
Bos Taurus to mouse
Mouse to human
13. AJF41183.1 and KC835294.1?
14. Orthologues
15. Orthologues
16. Mouse
17. Cytochrome oxidase
18. Influenza A virus (A/New York/492/2003(H1N2)) segment 4, complete sequence & Influenza
A virus (A/New York/492/2003(H1N2)) segment 6, complete sequence
19. Influenza A virus (A/New York/492/2003(H1N2)) for both
20. hemagglutinin & neuraminidase?
21. Reverse complement
22. 98%
23. 100%
24. T811 to G
25. Non conserved
Bioinformatics (2 points/ question for a total of 30 points)
1. Mus musculus
2. XM_023243939.1
3. 3e-127
4. orexin
5. 130 aa
6. Prepro-orexin
7. 2861 + 329 and 3015 +175
8. B
9. 1894 bp fragment
10. Capsid protein
11. NM_001037509.1
12. orthologues
13. 54.93%
14. Non conserved
15. reverse sequence
Assignment #4
Part I (2 points/ question for a total of 30 points) 1.
A. The gene you are interested in is not expressed in brain tissue.
2. 5.
3. Non-template strand
4. TTCAGG
5. 500
6. 5000
7. 1250
8. 100
9. A, B and D
10. C
11. B
12. D
13. B
14.
C. An RNA dependent RNA polymerase
D. An RNA dependent DNA polymerase
15. 5’GTGTCC 3’
Part II: Gene expression - Transcription (4.5 points/ question for a total of 45 points)
1. Indicate the ABS260, ABS280, the ABS260/ABS280 ratio and total yield of your RNA preparation.
2 points for ratio between 1.9-2.0
2.5 points for yield above 1µg/µL
2. Submit a figure of your RNA gel generated in lab exercise 6. Include an appropriate figure
legend.
Criteria for grading gel:
Figure provided with Accompanying legend
Figure title provided
Lanes are labelled and easy to understand
Legends should provide enough information so that the figure is understandable
and the experiment can be reproduced by someone who is familiar with molecular
biology.
3. Submit a table of the densitometric analysis of this northern hybridization. Your table must
include the raw data (the values for each of the areas) for actin and YRA, the normalized values
(YRA reading/actin reading) for each of the growth conditions and the relative expression as
compared to growth in the absence of osmotic shock. Give points if done appropriately
4. Submit a figure of the RT-PCR gel 1 generated in lab exercise 7. Include an appropriate figure
legend, which indicates the sizes of the products observed in each lane.
Criteria for grading gel:
Figure provided with Accompanying legend
Figure title provided
MW sizes of standard are indicated
Lanes are labelled and easy to understand
First lane is mw ladder
Legends should provide enough information so that the figure is understandable and the
experiment can be reproduced by someone who is familiar with molecular biology. Size
of amplicons is indicated.
Gel clearly shows bands and migration is satisfactory
Amplification product obtained where expected (see example below)
5. Remove genomic DNA?
6. Verify presence of genomic DNA
M 1 2 3 4 5
Reaction Template
PCR-1 R (Step I)
PCR-2 DR (step II)
PCR-3 RT-R (step III)
PCR-4 RT-DR (step III)
PCR-5 Yeast genomic DNA
2 p
oin
ts
2.5 points
7. RT dependent (approx. 1.4Kb and 450 bp) and RT independent (approx. 1.4 kb)
8. Presence of an intron of approximately 900 bases
9. Submit a figure of the RT-PCR gel 2 generated in lab exercise 7. Include an appropriate figure
legend, which indicates the sizes of the products observed in each lane.
Criteria for grading gel:
Figure provided with Accompanying legend
Figure title provided
MW sizes of standard are indicated
Lanes are labelled and easy to understand
First lane is mw ladder
Legends should provide enough information so that the figure is understandable and the
experiment can be reproduced by someone who is familiar with molecular biology. Sizes
of amplicons are indicated.
Size of Amplification product obtained are indicated
10. Submit a table of the densitometric analysis of these RT-reactions. Your table must include the
raw data (the values for each of the areas) for the largest rRNA band observed for the
corresponding condition on the corresponding ethidium bromide stained RNA gel (week 6)
and the corresponding YRA RT-PCR product observed on the RT-PCR gel 2, the normalized
values (YRA reading/rRNA reading) for each of the growth conditions and the relative
expression as compared to growth in the absence of osmotic shock. Give points if done
appropriately
Assignment #5
Part I 1. UAGGCU
2. MLFS
3. Same
4. Shorter
5. Similar
6. 34-35 b
7. 2690
8. 1890
9. 399 or 400
10. 1890
11. 215
12.
Fred and Helen’s conceived child Child ___2_
George and Helen’s conceived child Child ___3_
George and Helen’s adopted child Child ___1_
13. 1, 2 and 4
14. C3
15. 13
16. 3
17. BC
18. A, and D
19. D
20. 4 or 5
21. A. yfg1 and B
22. No effect
23. Decrease
24. Increase
25. Approx. 64%
Recommended