Lab 2: Finish Project 1- Autosomal or Sex-linked Inheritance?
- Examine each plate of F2 progeny. If you chose only L4 hermaphrodites, as instructed,you should only see hermaphrodite progeny. If you have a lot of males on your plates, you probably chose young adult worms rather than L4 hermaphrodite's. That's a problem - see your instructor.
- For each cross, you should count and examine a random sample of 100 worms. The mutant worms may be smaller and not move as well as the wild type worms. Look around your plate to get a quick assessment of the population.
- Record in your lab notebook the number of WT, Dpy, Unc, or Dpy Unc mutants by examining the phenotype as you remove each animal from the plate. (Be sure to flame the pick after removing each worm!!!)
If the genes responsible for the mutations are unlinked, you should see WT's (+/+;+/+), Dpy’s (dpy/dpy;+/+), Unc’s(+/+;unc/unc) and Dpy Unc’s (d/d;u/u) in a ratio of 9 WT:3 Dpy:3 Unc:1 Dpy Unc. If linked, you should see a greater proportion than expected of Dpy Unc’s (d u/d u) double mutants vs Dpy or Unc single mutants among the mutant hermaphrodite progeny.
The main challenge is to correctly differentiate single mutants of each phenotype (Dpy or Unc) from double mutants (Dpy AND Unc). Students tend to undercount single mutants and overcount doubles when they are inexperienced. Check with your instructor if you are unsure about how to score these groups. WT worms may be undercounted because they are harder to "catch". Be careful not to skew your data in this way. Pick what you consider single mutants to a plate and what you consider double mutants to another plate. Ask your instructor to check them before you go to far in the scoring process.
Make sure you copy your data into the appropriate place on the course spreadsheet!
You should now be able make educated conclusions about the inheritance of dpy and unc genes in these strains.
The three possible outcomes are:
- Autosomal and linked to a single chromosome
- Autosomal and unlinked
- One autosomal mutation and an x-linked mutation responsible for either the Dpy or Unc phenotype (which one?).
Lab 2: Start Forward Genetics Project 2: Testing a Mutant for True Breeding
To Do Today (per group)
- Choose one of the six plates of "mutagenized" worms at the front of the room
- Scan the “mutagenized” worms on this plate and determine their phenotype. Make sure you write this down.
- Transfer 1 putative mutant hermaphrodite to each of two new plates. Take care not to transfer any other animals or eggs other than your putative mutant.
- Label these 2 identical plates with the hermaphrodite mutant's strain information and your group's information (team color, initials, lab day, date). Use your BLUE Sharpie for labeling these linkage analysis plates. Labels belong on the side of the plate with the media in it. Tape makes it hard to see the worms on later days. DO NOT write on the lids - they fall off and you will not know what cover goes to what bottom!
- Put an elastic around the two phenotype determination plates. Place them in your group's plastic box (make sure your box is labeled with your names and lab section on a piece of your team color tape).
- Place your box on the shelf in the incubator for your lab section.
- Incubate your worms at 23°C for 3 days.
3 Days After Lab
- Examine your two plates containing your mutant worms and their F1 progeny. Are all the worms on at least one plate of the Dpy phenotype? If YES, then you are good to go on with the next cross. If you don't have all dumpy mutants in the F1 generation on a plate, you should NOT use that plate to select worms for the next cross. If neither of your plates contains all dumpy mutants, email your instructor ASAP!
- If you have an appropriate plate for continuing: Set up a pair of replicate crosses between 4 Dpy L4 hermaphrodites (should still have the vulval clearing about 1/2 way down the body of the worm) and 3-4 wild type male worms. You will have to search to find the wild type male worms among the hermaphrodites in the wild type worm plates provided in your lab day's box at the back of the room. When you are finished, you should have two identical mating plates with 3-4 L4 hermaphrodites and 3-4 wild type males.
- Label these identical plates with the hermaphrodite mutant's strain information and your group's information including the date. Use your BLUE Sharpie for labeling these linkage analysis/mapping plates.
- Incubate your worms at 23°C until next lab period.
Read Background information on your Series 2 Forward Genetics Project at Forward Genetics Project Summary. Submit statement of Series 2 overall experimental question & goals. Write a CONCISE one paragraph narrative summary of the investigation. Diagram the expected results (genotype and phenotype) for the crosses in the Linkage Testing work. Use only part (through Linkage 2) of the Project 2 cross template provided. (The Mapping part of the template will be completed later). Include a narrative description of the linkage testing experimental design and explain what you will look for in the results and why. Find a rubric and more information about this assignment at: Assignment Series 2 Summary & Linkage Testing Description. Due at the beginning of Lab 3.
See | Assignment_Help-_Data_Analysis_1 for instructions on Series 1 Results section write up for this project. Due at the beginning of Lab 4.