Monday Feb. 1 Use sterile hoods Make sure autoclave is on and reserved for the class time Stock solutions: Prepare the following stock solutions, all dissolved in 50% ethanol. Make up the first solution in each table, then make up the following by serial dilution as shown in the table using 1.5 mL microcentrifuge tubes. Then aliquot 100 µL into 0.5 mL microcentrifuge tubes so that each group will have a set of the tubes of each auxin that they will need. (NAA and IAA made up fresh; protect IAA from light; concentrations 10,000X those given in the lab manual on pp. 32-33 and reproduced in tables below), and 1 mL of 50% ethanol as control (keep these by hoods) 1-NAA Desired final concentrations Required stock solution Add to 5 x 10-7 M 5 x 10-3 M See #3 at beginning of TA prep 1.5 mL of 50% ethanol, adjust to give correct concentration 3 x 10-7 M 3 x 10-3 M 0.84 mL of 5 x M 0.56 mL of 50% ethanol 1.5 x 10-7 M 1-NAA 1.5 x M 0.7 mL of 3 x 10-3 M 0.7 mL of 50% ethanol 0.5 x 10-7 M 1-NAA 0.5 x M 0.47 mL of 1.5 x 10-3 M 0.93 mL of 50% ethanol 0 auxin 50% ethanol -- -- 2,4-D Desired final concentrations Required solution Add to 5 x 10-7 M 2,4-D 5 x 10-3 M 2,4-D See #3 at beginning of TA prep 1.5 mL of 50% ethanol, adjust to give correct concentration 3 x 10-7 M 2,4-D 3 x 10-3 M 2,4-D 0.84 mL of 5 x M 0.56 mL of 50% ethanol 1.5 x 10-7 M 2,4-D 1.5 x M 0.7 mL of 3 x 10-3 M 0.7 mL of 50% ethanol 3 3 3
Week 5 [Crosses with mutants; check plants & crosses] Set up PCRs Week 6 [Crosses with mutants; check plants & crosses] Run gels on PCRs Week 7 [Check plants & crosses; stop watering plants if appropriate] Discuss results, plan next experiments; set up PCRs Week 8 Presentations of projects [Collect crosses if ready] [Continue mapping] Week 9 [Collect crosses if ready; harvest mutant seed if ready] Discuss results, identify markers [Continue mapping] Week 10 Sterilize, begin characterization of mutants Continue mapping 1st lab report due Weeks 11-13 Characterization of mutants Continue mapping Week 14 Poster presentation Finish mapping & analyze results Week 15 Lab notebook due Final lab report due Schedule of 2nd example of genetics project lab (starting with existing mutants, parts B+C above)
Classical genetics project Molecular genetics project Week 1 Introduction Review of micropipettor use Sterilize control and F2 seeds for analysis of inheritance; spread on plates Begin literature review Week 2 [One day before lab: Put plates into growth chamber] Continue literature review Introduction Sterilize Col, Ler, mutant, and F3 seeds; spread on plates Week Analyze inheritance using F2 seeds Plant F2 seedlings Continue literature review Plan project: discuss with instructor [One day before lab: Put plates into growth chamber] Prepare Col and Ler DNA (from genetics project seedlings) Week 4 Revise project and prepare presentation Analyze F3 seedlings Prepare F3 DNA Week 5 Present project to class Begin experiments Set up 1st (control) PCR
Week 6 Present project to class Begin experiments Gel electrophoresis of 1st PCR Set up 2nd PCR (using F3 & control DNA) Week 7 Experiments Mapping, continued: PCR and electrophoresis Week 8 Experiments Mapping, continued: PCR and electrophoresis Week 9 Experiments Discuss progress with instructor 1st lab report due on mapping project Choose additional markers if feasible Week 10 Experiments Continue mapping Week 11 Experiments Analysis of results Prepare poster Continue mapping Week 12 Analysis of results Prepare poster Continue mapping if necessary Analysis of results Week 13 Analysis of results Week 14 Presentation of results (poster session) Week 15 Final lab reports due Lab notebooks due
2. Adapting the given lab to a once-a-week schedule The suggested schedule below essentially follows the schedule of the second example above. Because it would be impossible to fit in all four of the originally-planned experiments into a once-a-week schedule, we suggest that the instructor should choose either to carry out the analysis of inheritance (experiment 1) or of an aspect of the phenotype (experiment 2). An alternative schedule that more closely follows the original schedule is to delay the start of the molecular genetics project until the independent project is finished or almost finished, to enable students to concentrate on their independent project. Dates Classical genetics project (Expt 1 or 2, and Expt 3) Molecular genetics project (Expt 4) Week 1 Introduction Review of micropipettor use Sterilize control and experimental seeds for analysis of inheritance or phenotype; spread on plates Begin literature review Week 2 [One day before lab: Put plates into growth chamber] Continue literature review Introduction Sterilize Col, Ler, mutant, and F2 or F3 seeds; spread on plates Week Analyze inheritance using F2 seeds or [One day before lab: Put plates into
analyze phenotype Continue literature review Plan project: discuss with instructor growth chamber] Prepare Col and Ler DNA (from genetics project seedlings) Week 4 Revise project and prepare presentation Analyze F2 or F3 seedlings Prepare F2 or F3 DNA Week 5 Present project to class Begin experiments Set up 1st (control) PCR Week 6 Present project to class Begin experiments Gel electrophoresis of 1st PCR Set up 2nd PCR (using F3 & control DNA) Week 7 Experiments Mapping, continued: PCR and electrophoresis Week 8 Experiments Mapping, continued: PCR and electrophoresis Week 9 Experiments Discuss progress with instructor 1st lab report due on mapping project Choose additional markers if feasible Week 10 Experiments Continue mapping Week 11 Experiments Analysis of results Prepare poster Continue mapping