BISC219/F13: RNAi Lab 10

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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.

  1. Bring your 4 plates of CL2070 worms down to the microscope room.
  2. 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 what you see. Are the worms glowing? What part of the worms are glowing?

While some groups are examining their worms under the fluorescent scope you can score your WT and rrf-3 worms for different phenotypes.

Phenotype Definition
Unc uncoordinated
Slu sluggish
Prl paralyzed
Rol roller - U shaped roll instead of normal movement
Emb embryonic lethal - lots of dead eggs
Lvl larval lethal - lots of L1, L2, L3 larva that never grow up
Dpy Dumpy - short and stubby
Bmd body morphogenesis defect
Lon Long - more than normal length worms
Sma Small - short but not stubby like Dpy
Clr Clear - missing the full ovary in the adult hermaphrodite
Gro Growth defective - slow growing
Pvul Protruding vulva
Him 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.