BISC219/F11: RNAi Lab 10

Series 3 Reverse Genetics: Scoring RNAi Worms
3 days before next lab:
 * 1) Come into lab and find your stack of plates.
 * 2) On 2 of the experimental plates add 2 L4 wild type (N2) hermaphrodites
 * 3) On 2 of the experimental plates add 2 L4 rrf-3 hermaphrodites
 * 4) On 1 of the control plates add 2 L4 wild type (N2) hermaphrodites
 * 5) On 1 of the control plates add 2 L4 rrf-3 hermaphrodites
 * 6) Wrap all of your plates in an elastic and stick in your lab day box in the worm incubator set at 23°C

Phenotypic Analysis
Today in lab you will examine your RNAi worms and their progeny.
 * 1) Examine worms with a known bli-1 mutation to see what knockdown looks like.
 * 2) Compare your RNAi worms to worms that have a known bli-1 mutation - how do they compare?
 * 3) Examine your control worms to review wild type phenotype in comparison to mutants
 * 4) Compare your RNAi worms to the control worms - are they the same phenotype? Different? What do the “control” worms tell you?
 * 5) Examine your RNAi’d wild type (N2) and then your RNAi’d rrf-3 worms. Do you see any differences between the two treated strains of worms?

Be sure to record all of your results in your lab notebook. Take pictures of your control and RNAi worms to use in the results section of your next paper.

Assignment
Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab.

Outline of Experimental Design for REVERSE Genetics Project
Where are you now in this process? (What have you done so far? What's next?) A. Make the feeder strain of bacteria B. Plate wild type C. elegans worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest).  C. Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.
 * 1) Amplify gene of interest by PCR 
 * 2) Restriction Enzyme digestion of amplified DNA to create "sticky ends" for ligation
 * 3) Clean up DNA (remove enzymes) 
 * 4) Cloning: ligate gene into vector plasmid with amp resistance gene 
 * 5) Transform competent bacterial cells
 * 6) Select for transformants on media with ampicillin
 * 7) Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interest
 * 8) Culture the selected colony from colony pcr to create a lot of copies of these bacteria
 * 9) Isolate the cloned plasmid DNA from that cultured colony by miniprep
 * 10) Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA
 * 11) Select for transformants on media with ampicillin
 * 12) Choose an isolated colony to culture and make lots of feeder strain bacteria
 * 13) Induce expression of C. elegans gene dsRNA from the pL4440 vector in the bacteria by IPTG induction
 * 14) Seed NM lite worm growth media plates with feeder strain produced as described 

Links to Labs& Project Info
Series1: Worm Info Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start Lab 2: Sex-Linked or Autosomal Finale Series2: Background: Classical Forward Genetics and Gene Mapping Lab 2: Mutant Hunt Lab 3: Linkage Test Part 1 Lab 4: Linkage Test Part 2, Mapping and Complementation Lab 5: Finish Complementation; Mapping Continued Lab 6: DNA sequence analysis; Mapping Continued Lab 7: Complete Mapping: Score Series3:<BR> RNA interference<BR> RNAi General Information Media Recipes Lab 5: Picking your gene to RNAi Lab 6: Cloning your gene of interest Lab 7: Picking your transformant Lab 8: Plasmid purification and transformation Lab 9: Induction of bacteria for RNAi Lab 10: Scoring your worms Lab 11: