Genetics Virtual Lab book PDF

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This is the lab book for the genetics portion of the course...

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Biology 2290F Genetics Virtual Lab Book Sean Peters Section 001

Table of Contents Contacts…………………………………………………………………………………………………………………... Page 1 Restriction Digest Protocol………………………………………………………………………………………..Pages 2-3 Ligation Protocol……………………………………………………………………………………………………… Pages 4-5 Transformation Protocol…………………………………………………………………………………………. Pages 6-7 Transformation and Ligation Efficiency Results……………………………………………………….. Page 8 Miniprep Protocol…………………………………………………………………………………………………… Page 9-10 Individual Sequence………………………………………………………………………………………………… Page 11 Independent Experiment Protocol………………………………………………………………………….. Pages 12-17 Experimental Diagram ……………………………………………………………………………………………. Page 18 Expected Results Table …………………………………………………………………………………………… Page 19

September 14 2020 Contacts Instructor: Dr. Michelle Bolton [email protected] Teaching Assistant: Omar [email protected] Colleagues: Nandini Vyas [email protected] Phone Number: 6477612788 Seo Young Kim [email protected] Phone Number: 5198782304

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September 14 2020 Restriction Digest Protocol Kit name: FastDigest, From: Thermofisher Notes: The dependent variable is the type of single digest restriction enzyme (either Smal or EcoRI). These enzymes will be cutting the pUC18 plasmid in the multiple cloning site (LacZ operon), leaving it with either a blunt (Smal) or sticky (EcoRI) end. 1. Get five 1.5 mL microcentrifuge tubes and label them “Insert 1RE”, “Insert 2RE”, “H20 1RE”, “H20 2RE” and “TE” and your initials. 2. Look at the three tables below and add the appropriate components to each tube: a. You will first have to calculate the volume of plasmid to add to achieve 50 ng. b. Table 1. Components of pUC 18 plasmid with a concentration of 25 ng/ µL and restriction digests containing EcoRI, Smal, or TE Component: Insert tube, Blunt Volume Autoclaved dH2O 4 µL 5x FastDigest Buffer 2 µL 50 ng Plasmid DNA pUC18 (25 ng/ µL ) 2 µL SmaI **** 2 µL TOTAL 10 µL Component: Insert tube, Sticky Autoclaved dH2O 5x FastDigest Buffer 50 ng Plasmid DNA pUC18 (25 ng/ µL ) EcoRI**** TOTAL

Volume 4 µL 2 µL 2 µL 2 µL 10 µL

Component: H20 tube, Blunt Autoclaved dH2O 5x FastDigest Buffer 50 ng Plasmid DNA pUC18 (25 ng/ µL ) SmaI TOTAL

Volume 4 µL 2 µL 2 µL 2 µL 10 µL

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September 14 2020

Component: H20 tube, Sticky Autoclaved dH2O 5x FastDigest Buffer 50 ng Plasmid DNA pUC18 (25 ng/ µL ) EcoRI TOTAL

Volume 4 µL 2 µL 2 µL 2 µL 10 µL

Component: TE tube Autoclaved dH2O 5x FastDigest Buffer TE buffer EcoRI TOTAL

Volume 4 µL 2 µL 2 µL 2 µL 10 µL

3. Gently mix the tube by pipetting up and down. If there is liquid on the sides of the tube give it a quick spin in the centrifuge. 4. Place tube in 37 °C water bath for 30 min. 5. Once done, continue to Ligation Protocol

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September 14 2020 Ligation Protocol Kit name: T4 DNA ligase, From: Thermofisher Notes: The insert (R1) used was digested using the same restriction enzyme as the plasmid, the volume of insert used was calculated using the insert:vector ratio, and the three tubes without any insert are being used as controls 1. You are performing a ligation reaction with an insert:vector of 3:1. You will need to calculate the volume of insert to add to your tube. The ng of vector (plasmid) is kept constant at 50 ng. 2. To your restriction digest tubes, add the following: (ligase is in ice bucket at front of bench) Table 2. For the samples of interest components include ligation buffer, plasmid DNA, insert DNA R1, Water, and T4 DNA ligase. For the controls, all the components except for the insert DNA was included Ligation with insert +Blunt Component 5X Ligase reaction buffer Plasmid DNA (in tube already) Insert DNA R1 Autoclaved, dH2O T4 DNA ligase TOTAL

Volume 4 µL 10 µL 1 µL 3 µL 2 µL 20 µL

Ligation with insert +Sticky Component 5X Ligase reaction buffer Plasmid DNA (in tube already) Insert DNA R1 Autoclaved, dH2O T4 DNA ligase TOTAL

Volume 4 µL 10 µL 1 µL 3µL 2 µL 20 µL

Ligation with H20+Blunt Component 5X Ligase reaction buffer Plasmid DNA (in tube already)

Volume 4 µL 10 µL

Autoclaved, dH2O T4 DNA ligase TOTAL

4 µL 2 µL 20 µL

Ligation with H20+Sticky Component 5X Ligase reaction buffer Plasmid DNA (in tube already)

Volume 4 µL 10 µL

Autoclaved, dH2O T4 DNA ligase TOTAL

4 µL 2 µL 20 µL

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September 14 2020 Ligation with TE Component 5X Ligase reaction buffer TE buffer (in tube already)

Volume 4 µL 10 µL

Autoclaved, dH2O T4 DNA ligase TOTAL

4 µL 2 µL 20 µL

3. Gently mix by pipetting up and down and quick spin in the. Centrifuge if necessary. 4. Incubate both tubes at room temperature for 10 min (In the tube rack on your bench). 5. Take the tube of plasmid out of the styrofoam tube holder and place it in your ice bucket. Return the styrofoam tube rack with the tubes in it to the bucket near the competent cells. 6. Once done, continue to Transformation Protocol

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September 14 2020 Transformation Protocol (Not a kit) Notes: The test tubes with the insert are the samples of interest. The two labeled with H2O are controls used to ensure ligation is happening properly, and the one labeled TE are controls to ensure transformation is happening properly 1. Collect 6 tubes of competent E.coli JM101 (200 µL in each tube) in your ice bucket. ALL steps must be performed on ice. Label one tube” plasmid”, one tube “TE”, one tube “insert Blunt”, “insert Sticky” and one tube” H20 Blunt” and “H20 Sticky”. Put your initials on all tubes. 2. Add 10 µL of ligation with insert (Blunt or Sticky respectively) to the tube of cells labelled insert (Blunt or Sticky) 3. Add 10 µL of ligation with H20 (Blunt or Sticky respectively) to the tube of cells labelled H20 (Blunt or Sticky) 4. Add 10 µL of ligation with TE buffer to the tube of cells labelled TE 5. Add 2 µL p229 to the tube of cells labelled plasmid. 6. Mix gently by flicking the tube. 7. Incubate on ice for 5 min. 8. Incubate in 45 °C water bath for 100 s 9. Incubate on ice for 1 min 10. Add 250 µL LB broth to each tube 11. Incubate in 37 °C water bath for 15 min. 12. After 15 min incubation tubes can be kept at room temperature. 13. Gently mix your tubes by flicking or pipetting up and down to make sure cells have not settled. 14. Using your TE tube, make a 10-6 dilution. Dilute using the 0.5% NaCl “dilution solution”. Once your dilution is complete, keep the original buffer tube and your 106 dilution tube. All other tubes can be disposed of (10-2, 10-4).

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September 14 2020 15. You should now have 7 tubes: Insert Blunt, Insert Sticky, H20 Blunt, H20 Stick, Plasmid, TE and 10-6 dilution of TE. 16. Tidy your work area so nothing flammable is out. Collect four LB+100 mg/mL ampicillin plates and one LB plate. Label them around the circumference with your name, section number and what is on the plate (see step 25 for this information). 17. Light your Bunsen burner 18. Using sterile technique, one plate at a time, add 100 µL of: a. b. c. d. e.

Insert (Blunt and Sticky) on separate LB + amp plates H20 (Blunt and Sticky) on separate LB + amp plates Plasmid on LB + amp plate TE on LB + amp plate 10-6 dilution of TE on LB plate

19. Gather your 7 plates and put an elastic around them. 20. Place them in the wire rack upside down (label side up). 21. They will be incubated overnight at 37 °C. 22. Make sure all your enzymes are all in ice, all of your transformation, ligation and restriction digest tubes are in the waste containers. Keep the TE buffer tube in your tube rack. Once both partners are done, empty the waste containers in the orange bags at the front of the room. 23. Make sure your pipettes are returned to the green basket 24. Lysol your bench 25. Wash your hands.

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September 16 2020 Transformation and Ligation Efficiency Results Table 3: Shows the Transformation and Ligation Efficiency for the restriction enzymes Smal(Blunt end) and EcoRI (sticky end) used in the experiment for section 001 Blunt end Digest Sticky end Digest Transformation Ligation Transformation Efficiency Efficiency Efficiency 2.30E-08 0.15 1.03E-05 4.06E-06 0.26 8.88E-06 1.14E-05 0.33 1.97E-06 3.48E-05 0.35 1.27E-05 1.09E-05 0.14 1.39E-06 2.25E-05 0.13 1.60E-07 6.92E-06 0.29 1.29E-07 2.23E-06 0.33 3.35E-07 2.27E-06 0.22 1.54E-06 9.40E-08 0.31 6.52E-07 3.99E-06 0.14 3.79E-06 6.75E-06 0.11 3.20E-06 3.80E-06 0.28 3.56E-06 2.23E-06 0.19 5.33E-07 4.56E-07 0.18 3.55E-07 2.22E-06 0.28 2.46E-07 7.98E-05 0.23 1.07E-07 3.61E-06 0.15 4.99E-06 9.55E-07 0.31 3.10E-05 1.02E-05 0.29 8.22E-06

Ligation Efficiency 0.56 0.26 0.67 0.51 0.62 0.73 0.66 0.48 0.6 0.46 0.23 0.52 0.56 0.44 0.47 0.62 0.48 0.68 0.37 0.4

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September 16 2020 Mini-Prep Protocol Geneaid High-speed Plasmid mini kit 1. Collect your liquid culture that your prepared yesterday. 2. Transfer 1 mL of liquid culture to a 1.5 mL microcentrifuge 3. Centrifuge at 16 000 x g for 1 minute (wait until centrifuge is full/balanced) 4. Pour off the supernatant into the waste bucket 5. Transfer the final 1 mL of liquid culture to the same 1.5 mL microcentrifuge tube. 6. Centrifuge at 16 000 x g for 1 minute 7. Pour off the supernatant into the waste bucket 8. Add 200 µL of PD1 buffer to the tube 9. Resuspend pellet by vortexing 10. Add 200 µL of PD2 buffer 11. Mix gently by inverting the tube 10 times (DO NOT vortex) 12. Let stand at room temperature for 2 min. 13. Add 300 µL of PD3 buffer and mix immediately by inverting the tube 10 times 14. Centrifuge at 16 000 x g for 3 minutes (work with others to fill the centrifuge as much as possible – ensure centrifuge is balanced) 15. Locate the PD column sitting in the collection tube (orange tube in your tube rack) 16. Add the supernatant from step 14 to the column using a P200 pipette 17. Centrifuge at 16 000 x g for 30 s and discard (pour out) flow-through in waste bucket 18. Place PD column back in collection tube 19. Add 400 µL of W1 buffer to the column 20. Centrifuge at 16 000 x g for 30 s and discard flow through in waste bucket 21. Place PD column back in collection tube 22. Add 600 µL of wash buffer to the column 23. Centrifuge at 16 000 x g for 30 s and discard the flow-through in waste bucket 24. Place PD column back in collection tube 25. Centrifuge empty column at 16 000 x g for 3 min to dry column

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September 16 2020

26. Place PD column in new 1.5 mL centrifuge tube 27. Add 30 µL of elution buffer to the CENTRE of the PD column 28. Let stand for 2 min. 29. Centrifuge at 16 000 x g for 2 min to elute the purified DNA 30. Discard spin column, Keep and label the 1.5 mL centrifuge tube. 31. You will now need to Nanodrop your sample to determine the concentration a. Record the concentration and A260/A280 ratio in your lab book 32. Determine the volume of plasmid you require to provide 1 µg of DNA. Place this volume in a labelled tube and place it in the group tube rack. Fill out the information on the form.

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September 21 2020 My Individual Sequence 1. GTAAAACGACGGCCAGTGCCAAGCTTGCATGCCTGCAGGTCGACGTGTAGACGCCCGTGAAGG CCAAGGCGCTGAACAAGGAGGCGCTGCAGGCGGAGGTCGGGCTCCCGGTGGACCGGAACATC CCGGCCTGGGTGGCGTTCATCGGCAGGCTGGAAGAGCAGAAGGGACCCGACGTCATGGCGGC CGCCATCCCGCAGCTCATGGAGATGGTGGAGGACGTGCAGATCGTTCTGCTGGGCACGGGCAA GAAGAAGTTCGAGCGCATGCTCATGAGCGCCGAGGAGAAGTTCCCAGGCAAGGTGCGCGCCG TGGTCAAGTTCAACGCGGCGCTGGCGCACCACATCATGGCCGGCGCCGACGTGCTCGCCGTCA CCAGCCGCTTCGAGCCCTGCGGCCTCATCCAGCTGCAGGGGATGCGATACGGAACGCCCTGCG CCTGCGCGTCCACCGGTGGACTCGTCGATACCATCATCGAAGGCAAGACCGGGTTCCACATGG GCCGCCTCAGCTTCGACTGCAACGTCGTGGAGCCGGCGGACGTCAAGAAGGTGGCCACCACCT TGCAGCGCGCCATCAAGGTGGTCGGCACGCCGGCGTACGAGGAGATGGTGAGGAACTGCGCC GACGTGTCGGCGCCCGTGAAGGCCAAGGCGCTGAACAAGGAGGCGCTGCAGGCGGAGGTCG GGCTCCCGGTGGACCGGAACATCCCGGCCTGGGTGGCGTTCATCGGCAGGCTGGAAGAGCAG AAGGGACCCGACGTCATGGCGGCCGCCATCCCGCAGCTCATGGAGATGGTGGAGGACGTGCA GATCGTTCTGCTGGGCACGGGCAAGAAGAAGTTCGAGCGCATGCTCATGAGCGCCGAGGAGA AGTTCCCAGGCAAGGTGCGCGCCGTGGTCAAGTTCAACGCGGCGCTGGCGCACCACATCATGG CCGGCGCCGACGTGCTCGCCGTCACCAGCCGCTTCGAGCCCTGCGGCCTCATCCAGCTGCAGGG GATGCGATACGGAACGCCCTGCGCCTGCGCGTCCACCGGTGGACTCGTCGAAACCATCATCGA AGGCAAGACCGGGTTCCACATGGGCCGCCTCAGCGTCTACTGCAACGTCGTGGAGCCGGCGGA CGTCAAGAAGGTGGCCACCACCTTGCAGCGCGCCATCAAGGTGGTCGGCACGCCGGCGTACGA GGAGATGGTGAGGAACTGCGTCGAAGTGTCGATGCCCGTGAAGGCCAAGGCGCTGAACAAGG AGGCGCTGCAGGCGGAGGTCGGGCTCCCGGTGGACCGGAACATCCCGGCCTGGGTGGCGTTC ATCGGCAGGCTGGAAGAGCAGAAGGGACCCGACGTCATGGCGGCCGCCATCCCGCAGCTCAT GGAGATGGTGGAGGACGTGCAGATCGTTCTGCTGGGCACGGGCAAGAAGAAGTTCGAGCGCA TGCTCATGAGCGCCGAGGAGAAGTTCCCAGGCAAGGTGCGCGCCGTGGTCAAGTTCAACGCGG CGCTGGCGCACCACATCATGGCCGGCGCCGACGTGCTCGCCGTCAGAATTCGTAATCATGGTCA TAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACA The highlighted section is the M13 primer 2. A) The Latin name is Zea mays. B) The common name is maize; it is also known as corn. C) The gene name is LOC541854. D) The accession number is AAF79205.1. 3. A) The gene is on chromosome 9. B) Corn has 20 chromosomes; this is the diploid number. C) There are 14 exons. D) This gene encodes for the production of the granule-bound starch synthase 1 enzyme. This enzyme catalyzes a chemical reaction that biosynthesizes starch for the plant. Fujita, Naoko et al. “Function and characterization of starch synthase I using mutants in I using mutants in rice.” Plant physiology vol. 140,3 (2006): 1070-84. doi:10.1104/pp.105.071845

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September 23 2020 Independent Experiment Protocol Notes: This experiment will determine whether insert size has any impact on the transformation and ligation efficiency. A pUC18 plasmid will be cut using the EcoR1 restriction enzyme, and ligated with either a Sm insert or Lrg insert, also cut with a restriction enzyme. The plasmid will then be transformed into a bacteria and plated onto an LB+Ampicillin plate. Restriction Digest 1. Get five 1.5 mL microcentrifuge tubes and label them “Insert Sm”, “Insert Lrg”, “H20 ligation”, and “TE” and your initials. 2.Look at the three tables below and add the appropriate components to each tube: Table 4 Components of pUC 18 plasmid with a concentration of 25 ng/ µL and restriction digests containing EcoRI and TE Component: Insert tube (for Sm) Autoclaved dH2O 5x FastDigest Buffer 50 ng Plasmid DNA pUC18 (25 ng/ µL ) EcoRI TOTAL

Volume 4 µL 2 µL 2 µL 2 µL 10 µL

Component: Insert tube (for Lrg) Autoclaved dH2O 5x FastDigest Buffer 50 ng Plasmid DNA pUC18 (25 ng/ µL ) EcoRI TOTAL

Volume 4 µL 2 µL 2 µL 2 µL 10 µL

Component: H20 tube Autoclaved dH2O 5x FastDigest Buffer 50 ng Plasmid DNA pUC18 (25 ng/ µL ) EcoRI TOTAL

Volume 4 µL 2 µL 2 µL 2 µL 10 µL

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Component: TE tube Autoclaved dH2O 5x FastDigest Buffer TE buffer EcoRI TOTAL

Volume 4 µL 2 µL 2 µL 2 µL 10 µL

3. Gently mix the tube by pipetting up and down. If there is liquid on the sides of the tube give it a quick spin in the centrifuge. 4. Place tube in 37 °C water bath for 30 min. 5. You are performing a ligation reaction with an insert:vector of 3:1. You will need to calculate the volume of insert to add to your tube. The ng of vector (plasmid) is kept constant at 50 ng Ligation Table 5 For the samples of interest components include ligation buffer, plasmid DNA, insert DNA Sm or Lrg with a concentration of 20 ng/µL, Water, and T4 DNA ligase. For the controls, all the components except for the insert DNA was included Ligation with Sm Insert Component 5X Ligase reaction buffer Plasmid DNA (in tube already) Insert DNA Sm Autoclaved, dH2O T4 DNA ligase TOTAL

Ligation with Lrg Insert Volume 4 µL 10 µL 0.6 µL 3.4 µL 2 µL 20 µL

Component 5X Ligase reaction buffer Plasmid DNA (in tube already) Insert DNA Lrg Autoclaved, dH2O T4 DNA ligase TOTAL

Volume 4 µL 10 µL 2.8 µL 1.2 µL 2 µL 20 µL

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September 23 2020 Ligation with H2O, no insert

Ligation with TE

Component 5X Ligase reaction buffer Plasmid DNA (in tube already)

Volume 4 µL 10 µL

Autoclaved, dH2O T4 DNA ligase TOTAL

4 µL 2 µL 20 µL

Component 5X Ligase reaction buffer TE buffer (in tube already)

Volume 4 µL 10 µL

Autoclaved, dH2O T4 DNA ligase TOTAL

4 µL 2 µL 20 µL

6. Gently mix by pipetting up and down and quick spin in the. Centrifuge if necessary. 7. Incubate both tubes at room temperature for 10 min (In the tube rack on your bench). 8. Take the tube of plasmid out of the styrofoam tube holder and place it in your ice bucket. Return the styrofoam tube rack with the tubes in it to the bucket near the competent cells. Transformation 9. Collect 5 tubes of competent E.coli JM101 (200 µL in each tube) in your ice bucket. ALL steps must be performed on ice. Label one tube” plasmid”, one tube “TE”, one tube “insert Sm”, “insert Lrg” and one tube” H20”. Put your initials on all tubes. 10. Add 10 µL of ligation with insert (Sm or Lrg) to the tube of cells labelled insert (Sm or Lrg) 11. Add 10 µL of ligation with H20 to the tube of cells labelled H2O 12. Add 10 µL ligation with TE buffer to the tube of cells labelled TE 13. Add 2 µL pUC18 to the tube of cells labelled plasmid. 14. Mix gently by flicking the tube. 15. Incubate on ice for 5 min. 16. Incubate in 45 °C water bath for 100 s 17. Incubate on ice for 1 min

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September 23 2020 18. Add 250 µL LB broth to each tube 19. Incubate in 37 °C water bath for 15 min. 20. After 15 min incubation tubes can be kept at room temperature. 21. Gently mix your tubes by flicking or pipetting up and down to make sure cells have not settled. 22. Using your TE tube, make a 10-6 dilution. Dilute using the 0.5% NaCl “dilution solution”. Once your dilution is complete, keep the original buffer tube and your 10-6 dilution tube. All other tubes can be disposed of (10-2, 10-4). 23. You should now have 5 tubes: Insert Sm, Insert Lrg, H20, Plasmid, TE and 10-6 dilution of TE. 24. Tidy your work area so nothing flammable is out. Collect four LB+100 mg/mL ampicillin plates and one LB plate. Label them around the circumference with your name, section number and what is on the plate (see step 25 for this information). 25. Light your Bunsen burner 26. Using sterile technique, one plate at a time, add 100 µL of: a. b. c. d. e.

Insert (Sm and Lrg) on separate LB + amp plates H20 on separate LB + amp plates Plasmid on LB + amp plate TE on LB + amp plate 10-6 dilution of TE on LB plate

27. Gather your 5 plates and put an elastic around them. 28. Place them in the wire rack upside down (label side up). 29. They will be incubated overnight at 37 °C. 30. Make sure all your enzymes are all in ice, all of your transformation, ligation and restriction digest tubes are in the waste containers. Keep the TE buffer tube in your tube rack. Once both partners are done, empty the waste containers in the orange bags at the front of the room. 31. Make sure your pipettes are returned to the green basket 15

September 23 2020 32. Lysol your bench 33. Wash your hands. Miniprep 34. Collect your liquid culture that your prepared yesterday. 35. Transfer 1 mL of liquid culture to a 1.5 mL microcentrifuge 36. Centrifuge at 16 000 x g for 1 minute (wait until centrifuge is full/balanced) 37. Pour off the supernatant into the waste bucket 38. Transfer the final 1 mL of liquid culture to the same 1.5 mL microcentrifuge tube. 39. Centrifuge at 16 000 x g for 1 minute 40. Pour off the supernatant into the waste bucket 41. Add 200 µL of PD1 buffer to the tube 42. Resuspend pellet by vortexing 43. Add 200 µL of PD2 buffer 44. Mix gently by inverting the tube 10 times (DO NOT vortex) 45. Let stand at room temperature for 2 min. 46. Add 300 µL of PD3 buffer and mix immediately by inverting the tube 10 times 47. Centrifuge at 16 000 x g for 3 minutes (work with others to fill the centrifuge as much as possible – ensure centrifuge is balanced) 4...