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.

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Assignment 1Dilutions 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)

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1. 0.2M 6. 2.1M2. 1 part solute & 7 parts of solvant 7. 0.5M3. 6M 8. 2.3M4. 12 mL 9. 25.75. 1M 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)

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.

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1 4 6 10

B E B E

BamHI

BamHI

EcoRI

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)

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).

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10/0 Kbp

3 Kbp

6 Kbp

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:

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 → 0.55 → 0.5 + 4.56 → 0.5 + 5.5

5. 1 µg of 1Kbp4 µg of 2Kbp3 µg of 3Kbp4 µg of 4Kbp

6.Enzyme digest Fragment sizes

BamHI 3 and 7Kbp

EcoRI 10Kbp

BamHI + EcoRI 3 and 4Kbp

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EcoRI 8.9 kbBamHI 6 kb. 2.9 kbHindIII 8.9 kbEcoRI + BamHI 6 kb, 2.4 kb, 0.5 kbEcoRI + HindIII 7.4 kb, 1.5 kbBamHI + HindIII 5 kb, 2.9 kb, 1 kbBamHI + EcoRI + HindIII 5 kb, 2.4 kb, 1 kb, 0.5 kb

BamHI

HindIII

EcoRI

7. 10Kbp8. Only this map is possible.9. 3 → 1 + 2

7 → 6 + 110. Any rotation and mirror image of this map are valid.

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8.9/0 Kbp

2.4 Kbp

7.4 Kbp

BamHI8.4 Kbp

Lab exercise(4 points/question)

Dilution Exercise:1.

a. A 1.5mM solution of compound “A”. 0.12ml Soln. 1 + 0.88ml waterb. A 0.36% (m/v) solution of compound “B”. 0.05ml soln. 2 + 0.95ml waterc. A 6% (v/v) solution of solution I. 0.06ml soln 1 + 0.94ml waterd. 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 watere. A solution representing the following ratio: solution I: solution II : water : 2:1:2

0.4ml soln. 1, 0.2ml soln. 2 + 0.4ml water2. 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.

0.551b.

0.186c.

0.291d.

0.324

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.

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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.

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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 3.82, 0.18BamHI 4.0, 2.35, 1.65EcoRI 3.0, 1.0HindIII + BamHI 3.55, 2.35, 1.2, 0.27, 0.18HindIII + EcoRI 1.87, 1.0, 0.95, 0.18BamHI + EcoRI 1.6, 1.4, 1.0, 0.75, 0.25

2. What is the total plasmid size? 4Kbp3. 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? 4Kbp5. Draw a circular map which is in agreement with the results presented. (8 points)

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

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HindIII

1 2 3 4 5

16Kb

8 Kb

2.5 Kb2.0 Kb 2

1.0 Kb

0.5 Kb

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).

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

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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

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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:

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1. Mol wt. marker2. Uncut3. BamH14. HindIII5. PstI6. PvuII7. ScaI8. 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.

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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 Organism Definition Protein id. Gene productY00417 Triticum aestivum Wheat mitochondrial COI gene

for cytochrome oxidase subunit ICAA68474.1 cytochrome oxidase

subunit IX01108 Triticum aestivum Wheat mitochondrial cytochrome

oxidase subunit II (CO II) geneCAA25581.1 cytochrome oxidase

subunit IIAB022060 Triticum aestivum Triticum aestivum mitochondrial

gene for ORF25BAA82046.1 ORF25

X02352 Triticum aestivum Wheat mitochondrial gene for apocytochrome b

CAA26207.1 apocytochrome b

X15944 Triticum aestivum Triticum aestivum mitochondrial cox3 gene

CAA34071.1 cytochrome oxidase subunit III

Accession Coverage Max ident. E valueY00417 42% 100% 0X01108 25% 100% 0AB022060 18% 99% 0X02352 31% 99% 0X15944 30% 100% 0

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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.

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