BISC 219/2009:RNA interference

These labs were developed with the help of the Silencing Genomes project of the Dolan DNA Learning Center.

RNAi General Information
Media Recipes
Lab 4: Picking your gene to RNAi
Lab 5: Plasmid DNA isolation and transformation
Lab 6: Induction of RNAi plasmid and C. elegans feeding
Lab 7: Single Worm PCR and Agarose Gel Electrophoresis
Lab 8: PCR Reaction Cleanup and Sequencing

Schedule of Experiments

Lab #	Dates	Activity	Outside lab time
4	9/29 - 10/5	Pick a single bacterial colony containing a plasmid for RNAi	The night before next lab: Set up an overnight culture of your colony
5	10/6 - 10/14	Plasmid DNA isolation Transformation of the isolated plasmid into the HT115(DE) feeding strain	The next day: Check control and transformation plates for growth - save your transformation plate The night before next lab: Set up an overnight culture of a single colony from your transformation
6	10/19 - 10/23	Induction of the bacteria to produce RNA Seed plates and dry for bacterial feeding RNAi	4 days later: Pick 2 L4 hermaphrodite worms of N2 and rrf-3 genotype to 2 RNAi plates for each genotype and make 1 control plate for each genotype as well (6 plates total) Incubate at 23°C until next class
7	10/26 - 10/30	Examine the phenotypes of the fed worms - compare to control N2 worms and worms containing a mutation in the gene you are examining Single worm PCR of N2 and RNAi treated worms Agarose gel electrophorsis of PCR products
8	11/4 - 11/10	Clean up PCR reactions with Qiagen Min-Elute kit Run sequencing reaction Clean up sequencing reaction with columns	Analyze your sequencing data