Report exp 6 and 7 (DNA and RNA)

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Report exp 6 and 7 (DNA and RNA)

  1. 1. Experiment #6<br />NUCLEIC ACID - RNA<br />Group 7<br />BALDA<br />SANTOS<br />TALAG<br />VILLACORTA<br />
  2. 2. Objectives<br />To be able to isolate RNA from yeast<br />To be able to get the percentage by mass of RNA from yeast<br />To be able to identify products of hydrolysis of RNA <br />To perform tests for identification of products of RNA hydrolysis<br />
  3. 3. Introduction<br />RNA (ribonucleic acid)<br />Genetic material of certain viruses<br />Made up of a single strand of nucleotides<br />Directs the middle steps of protein production<br />Polymer of Purine and Pyrimidine ribonucleotides linked through 3’-5’ phosphodiester bridges <br />With Uracil and D-ribose<br />
  4. 4. Experiment Proper<br />6.2 Hydrolysis of RNA<br />Materials<br /><ul><li>Mortar and pestle
  5. 5. Erlenmeyer Flask
  6. 6. 0.2% NaOH
  7. 7. 10% NaOH
  8. 8. 0.5%CuSO4</li></li></ul><li>Hydrolysis<br />Chemical process involving the addition of water causing a molecule to cleave into two parts<br /> Phosphate<br /> Nucleic Acids<br /> Nucleoprotein<br />Nucleotides<br /> Protein<br />D-ribose<br />Pyrimidine<br />Purine<br />
  9. 9. Experiment Proper<br />6.3 Identification Tests<br />Materials<br /><ul><li>NaOH
  10. 10. CuSO4
  11. 11. 10% NH4OH
  12. 12. 2% AgNO3
  13. 13. Benedict’s Reagent
  14. 14. 0.2M Ammonium Molybdate</li></li></ul><li>Identification Tests<br />Biuret Test<br />-test to detect the presence of peptide bonds <br />Principle: Formation of purple colored product due to the reduction of Cu2+to Cu+ made by the detected reducing sugar<br />Reagents:NaOH and CuSO4<br />
  15. 15. Identification Tests<br />10% AgNO3 and 10% NH4OH<br />-test to detect the presence of Purine Bases <br />Principle: Hydrolysis of N-β-glucosidic bonds between purine bases and ribose or deoxyribose results in a release of purine bases(A and G) caused by NH4OH. Ag+ precipitate caused the formation of foamy gelatinous substance. <br />Reagents: AgNO3 and NH4OH<br />
  16. 16. Identification Tests<br />Benedicts Test<br />-test to detect the presence Reducing Sugars <br />Principle:Cu2+ react with reducing sugars = precipitate of cuprous oxide (Cu2O) produces a change in the Benedict’s reagent from blue to green or reddish-orange, depending on amount of reducing sugar. The cyclic sugar is reduced to linear aldehyde.<br />Green = small amn’t of reducing sugars<br />Red-orange = abundance of reducing sugars<br />Reagents:NaOH, CuSO4 and tartaric acid<br />
  17. 17. Identification Tests<br />Ammonium Molybdate Test<br />-test for Inorganic Phosphate <br />Principle: hydrolysis of pyrophosphate to phosphate forming yellow ppt.<br />Reagents: (NH4)6 Mo7O24 · 4H2O and H2SO4<br />
  18. 18. Experiment Proper<br />
  19. 19. Experiment Proper<br />Biuret’s Test<br />10% NH4OH & 10% AgNO3<br />Benedict’s Test<br />Ammonium Molybdate<br />
  20. 20. Ideal Results<br />
  21. 21. Comparison <br />
  22. 22. PLQ’s<br />What products of hydrolysis did the yeast RNA yield?<br /><ul><li>Nucleobases (A, G, U and C)
  23. 23. Sugar (D-ribose)
  24. 24. Phosphate</li></li></ul><li>PLQ’s<br />2. Cite qualitative tests to characterize the products of RNA hydrolysis. Are these specific for RNA?(explain)<br /><ul><li>Bial’s Test
  25. 25. Test for pentoses.
  26. 26. specimen is heated with sol’n of orcinol, HCl, and FeCl3
  27. 27. pentoses : dehydrated to furfural = reacts with orcinol to form a green product</li></li></ul><li>PLQ’s<br /><ul><li> Wheeler and Johnson's test </li></ul>- Treatment of Cytosine and Uracil w/ bromine water yields dialuric acid = green coloration<br /> - Addition of Ba(OH)2 = purple.<br />
  28. 28. PLQ’s<br /><ul><li>Murexide Test </li></ul> - Murexide (NH4C8H4N5O6, or C8H5N5O6.NH3) or ammonium purpurate [MX] = ammonium salt of purpuric acid.<br /> - Test for presence of uric acid = white, odorless, tasteless crystalline substance from purine degradation<br /> - Positive : yellow residue<br /> - A & G : purines = 2-ringed crystalline organic base – a uric acid; heterocyclic aromatic org. compound composed of pyrimidine ring fused w/ an imidazole ring<br />
  29. 29. PLQ’s<br />3. What are differences of RNA and DNA?<br />
  30. 30. Experiment #7<br />NUCLEIC ACID - DNA<br />Group 7<br />BALDA<br />SANTOS<br />TALAG<br />VILLACORTA<br />
  31. 31. Objectives<br />To be able to identify products of hydrolysis of DNA<br />To perform tests for identification of products of DNA hydrolysis<br />
  32. 32. Introduction<br />DNA (deoxyribonucleic acid)<br />Carries the Genetic Code of Life<br />Made up of a double strand of nucleotides in anti-parallel direction<br />With Thymine and 2-deoxy-D-ribose<br />Widely studied for medical diagnosis and production of therapeutic proteins <br />
  33. 33. Experiment Proper<br />7.1 Hydrolysis of DNA<br />Materials<br /><ul><li>10% NH4OH
  34. 34. 2% AgNO3
  35. 35. 5g NaCl
  36. 36. Ammonium Molybdate
  37. 37. Benedict’s Reagent
  38. 38. 0.2% NaOH
  39. 39. 10% NaOH
  40. 40. 0.5%CuSO4
  41. 41. Filter paper
  42. 42. Wire Loop</li></li></ul><li>Hydrolysis<br />Chemical process involving the addition of water causing a molecule to cleave into two parts<br /> Phosphate<br /> Nucleic Acids<br /> Nucleoprotein<br />Nucleotides<br /> Protein<br />2-deoxy D-ribose<br />Pyrimidine<br />Purine<br />
  43. 43. Experiment Proper<br />Materials<br /><ul><li>NaOH
  44. 44. CuSO4
  45. 45. 10% NH4OH
  46. 46. 2% AgNO3
  47. 47. Benedict’s Reagent
  48. 48. 0.2M Ammonium Molybdate</li></li></ul><li>Identification Tests<br />Biuret Test<br />-test to detect the presence of peptide bonds <br />Principle: Formation of purple colored product due to the reduction of Cu2+to Cu+ made by the detected reducing sugar<br />Reagents:NaOH and CuSO4<br />
  49. 49. Identification Tests<br />10% AgNO3 and 10% NH4OH<br />-test to detect the presence of Purine Bases <br />Principle: Hydrolysis of N- β -glucosidic bonds between purine bases and ribose or deoxyribose results in a release of purine bases(A and G) caused by NH4OH. Ag+ precipitate caused the formation of foamy gelatinous substance. <br />Reagents: AgNO3 and NH4OH<br />
  50. 50. Identification Tests<br />Benedicts Test<br />-test to detect the presence Reducing Sugars <br />Principle:Cu2+ react with reducing sugars = precipitate of cuprous oxide (Cu2O) produces a change in the Benedict’s reagent from blue to green or reddish-orange, depending on amount of reducing sugar. The cyclic sugar is reduced to linear aldehyde.<br />Green = small amn’t of reducing sugars<br />Red-orange = abundance of reducing sugars<br />Reagents:NaOH, CuSO4 and tartaric acid<br />
  51. 51. Identification Tests<br />Ammonium Molybdate Test<br />-test for Inorganic Phosphate <br />Principle: hydrolysis of pyrophosphate to phosphate forming yellow ppt.<br />Reagents: (NH4)6 Mo7O24 · 4H2O and H2SO4<br />
  52. 52. Results<br />
  53. 53. Experiment Proper<br />Biuret’s Test<br />10% NH4OH & 10% AgNO3<br />Benedict’s Test<br />Ammonium Molybdate<br />
  54. 54. Ideal Results<br />
  55. 55. Comparison <br />
  56. 56. PLQ’s<br />1. What are the reagents used to extract DNA and RNA?<br /><ul><li>RNA : NaOH and C2H5OH(ethanol) with Conc. HCl
  57. 57. DNA : NaCl, ethanol and detergent </li></li></ul><li>PLQ’s<br />2. Did you have the same results of qualitative tests performed in experiment 6? (Explain)<br /><ul><li>No, but reality check, the results in both experiments should be identical for they yield the same hydrolysate and the tests will yield the same products except for the Benedict’s test because the sugar that it will detect is 2-deoxy-D-ribose where in experiment 6, the sugar is D-ribose.</li></li></ul><li>PLQ’s<br />3. What tests specifically detect ribose and 2-deoxy ribose? Discuss the principle.<br /><ul><li>Based from the experiment, it is the Benedict’s Test.</li></ul>-Cu2+ react with reducing sugars = precipitate of cuprous oxide (Cu2O) produces a change in the Benedict’s reagent from blue to green or reddish-orange, depending on amount of reducing sugar. The cyclic sugar is reduced to linear aldehyde.<br />
  58. 58. PLQ’s<br />But…<br /><ul><li>Dische Diphenylamine Test
  59. 59. used to detect the presence of DNA. A positive test for DNA is indicated by a blue color change.
  60. 60. The reaction depends on the conversion of the pentose to ω-hydroxylaevulinicaldehyde which then reacts with diphenylamine to give a blue colored complex. The intensity of the blue color is proportional to the concentration of DNA. Dische reagent does not react with the ribose sugar in RNA and does not form a blue-colored complex</li></li></ul><li>PLQ’s<br /><ul><li>Bial’s Test
  61. 61. Test for pentoses.
  62. 62. specimen is heated with sol’n of orcinol, HCl, and FeCl3
  63. 63. pentoses : dehydrated to furfural = reacts with orcinol to form a green product</li></li></ul><li>PLQ’s<br />4. Can DNA be isolated from beef? Discuss the process briefly.<br />Yes!<br />breakage of cell -> membrane lipid removal -> protein removal -> RNA removal -> precipitation of DNA<br />

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