Lab 10: Phenotype Analysis of hsf-1 RNAi worms
Instructors will do before lab:
Your instructor or our lab specialist will come in 4-5 hours before lab and "heat shock" one plate of each of your RNAi samples: wild type treated, wild type control, rrf-3 treated, rrf-3 control, CL2070 treated and CL2070 control. The second plate of each condition serves as your heat shock control. Heat shocking involves moving the worms to a 37°C incubator for 60 minutes. The worms will then be placed back at their initial temperature until you use them tomorrow.
To Do in Lab Today
Reverse genetics allows us to start with a known gene sequence and determine the phenotype associated with it. You will determine the effect of RNAi of hsf-1 on several different strains of worms.
We only have one fluorescent compound scope to view the worms and one fluorescent dissecting scope. While some groups are scoring their wild type and rrf-3worms for phenotypes others will be working with the instructor in the microscope room to view and photograph their GFP worms.
- Bring your 4 plates of CL2070 worms down to the microscope room.
- You will look at your worms and take some pictures under the fluorescent dissecting scope. Place one of your non-heat shocked plate on the stage of the dissecting scope and expose the worms to the UV light. Record in your notebook the relative level of GPF expression (intensity and location of green fluorescence) in worms of each treatment. Are the worms green and glowing? What parts of the worms are glowing most?
In the lab, while some groups are examining their worms under the fluorescent scope, you can score your WT and rrf-3 worms for different phenotypes using your regular, white light, dissecting microscopes.
| Phenotype || Definition
|| roller - U shaped roll instead of normal movement
|| embryonic lethal - lots of dead eggs
|| larval lethal - lots of L1, L2, L3 larva that never grow up
|| Dumpy - short and stubby
|| body morphogenesis defect
|| Long - more than normal length worms
|| Small - short but not stubby like Dpy
|| Clear - missing the full ovary in the adult hermaphrodite
|| Growth defective - slow growing
|| Protruding vulva
|| High incidence of males - more than 10% of the population is male
Score by examining the worms and keeping track of how many of each phenotype you see. The table above is a short list of those possible. You may or may not see these phenotypes. Take pictures to document some of the phenotypes you see.
Please complete the graded Assignment described in BISC219/F13:Assignments due for all students on the class day of classes by 4pm.