Molecular Biology 2016 Assignment 1 Dilutions and Concentrations Only submit the answers to one digit after the decimal, you do not need to show your calculations (1 point/question) 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 LiCl? 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 an 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) Molecular Biology 2016 Restriction Enzymes & Restriction Mapping (3 points/question) 11. The nomenclature of restriction enzymes can provide useful information about the source of the enzyme. For example, EcoRI indicates that this enzyme was the first enzyme isolated from an E.coli strain “R”. From which bacteria was PvuII isolated from? 12. Define the following terms: Isoschizomer, neoschizomer, and isocaudomer. 13. Amongst the enzymes listed below, which if any, generate compatible ends to each other? (Ex. A and B) Enzyme Recognition Sequence Cut EcoRI 5'GAATTC 5'---G AATTC---3' BamHI 5'GGATCC 5'---G GATCC---3' HindIII 5'AAGCTT 5'---A AGCTT---3' TaqI 5'TCGA 5'---T CGA---3' NotI 5'GCGGCCGC 5'---GC GGCCGC---3' Sau3AI 5'GATC 5'--- GATC---3' PvuII 5'CAGCTG 5'---CAG CTG---3' KpnI 5'GGTACC 5'---GGTAC C---3' PstI 5'CTGCAG 5'---CTGCA G---3' SacI 5'GAGCTC 5'---GAGCT C---3' SalI 5'GTCGAC 5'---G TCGAC---3' ScaI 5'AGTACT 5'---AGT ACT---3' SpeI 5'ACTAGT 5'---A CTAGT---3' SphI 5'GCATGC 5'---GCATG C---3' StuI 5'AGGCCT 5'---AGG CCT---3' XbaI 5'TCTAGA 5'---T CTAGA---3' Molecular Biology 2016 14. 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 2Kbp and 2Kbp) B E B E 1 4 6 10 Enzyme digest Fragment sizes BamHI EcoRI BamHI + EcoRI 15. 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. (Fragments which contain one or more BamHI site which remains undigested) 16. Consider the results of the BamHI digest you indicated in the above table. Draw all possible maps, (including the one illusrated above) which could correspond to the results indicated for the BamHI digest alone. 17. It was determined that the enzyme XhoI cuts at 2.0Kbp 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 16. (Ex. 4Kbp BamHI → 3 + 1 Kbp). 18. 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. Molecular Biology 2016 19. 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 2Kbp and 2Kbp) 10/0 Kbp EcoRI BamHI 3 Kbp 6 Kbp BamHI Enzyme digest Fragment sizes BamHI EcoRI BamHI + EcoRI 20. 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. (Fragments which contain one or more BamHI site which remains undigested) 21. Consider that the EcoRI site is invariable. Is this the only possible map according to the results presented in the above table? If not draw another possible map. 22. 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 questions 19 and 21. (Ex. 4Kbp BamHI → 3 + 1 Kbp). Molecular Biology 2016 23. An 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 8.9 kb BamHI 6 kb. 2.9 kb HindIII 8.9 kb EcoRI + BamHI 6 kb, 2.4 kb, 0.5 kb EcoRI + HindIII 7.4 kb, 1.5 kb BamHI + HindIII 5 kb, 2.9 kb, 1 kb BamHI + EcoRI + HindIII 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. 24. Obtain the picture of the agarose gel electrophoresis of digests of the plasmid pBR322. (The file can be found on the course’s web site under the rubric “Sequences>pBR322”. Based on the results obtained, answer the following questions: a. How many times did PvuII cut within the plasmid? b. How many times did HincII cut within the plasmid? c. How many times did HincII cut within the PvuII fragment? d. What are the distances between the PvuII and the HincII sites? Molecular Biology 2016 Lab exercise (4 points/question) Dilutions exercise with micropipettors (Pg. 13) 1. Indicate the absorbance readings obtained for each of the following solutions which you prepared in the first lab exercise. If you are submitting the assignment as a group of 2, indicate the average absorbance for both samples. a. A 1.5mM solution of compound “A”. b. A 0.36% (m/v) solution of compound “B”. c. A 6% (v/v) solution of solution I. d. A solution containing 0.5mg of compound “A” and 0.1% (v/v) of compound “B”. e. A solution representing the following ratio: solution I: solution II : water : 2:1:2 Determining DNA concentration: (Pg. 14) 2. Submit a table of the DNA concentration determinations experiment performed on page 14 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. 3. Submit a graph representing the A260 readings Vs standard DNA concentrations. Include a line of best fit, the R coefficient, and the formula of the line. 4. Determine from your graph, what DNA concentration in µg/mL corresponds to an A260 of 1.0. 5. According to the constant determined in the previous question, what was the DNA concentration of the undiluted unknown DNA solution provided? Restriction digests & agarose gel electrophoresis (Pg. 15-17) 6. Submit a figure and an appropriate figure legend of the agarose gel described on page 17 of the lab manual. 7. Submit a standard curve of the molecular weight ladder (Migration distance Vs. Size in Kbp) 8. Submit a table of the restriction digests of the recombinant plasmid which includes the following information: Enzyme used, number of cuts, fragment sizes observed. 9. 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. 10. 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. (See directives on this course’s web site) Molecular Biology 2016 Bioinformatics 1 11. Submit a table with the following information with regards to each of the unknown genes from the first bioinformatics exercise.