BISC 219/F10: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 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 and RNA purification
Lab 11: RT PCR reactions

Schedule of Experiments

Lab #	Dates	Activity	Outside lab time
5	10/5 - 10/12	Pick a gene of interest Set up a PCR reaction to clone the gene	Examine the results of the agarose gel electrophoresis
6	10/13 - 10/19	Restriction enzyme digest of PCR product Cleanup and ligation into the pPD129.36 vector Transformation of the isolated plasmid into the BL21 cloning E. coli	The day after lab: Check control and transformation plates for growth - save your transformation plate
7	10/20 - 10/26	Colony PCR to check for transformation	The night before next lab: Set up an overnight culture of a single colony from your transformation
8	10/28 - 11/3	Plasmid isolation from the BL21 cells Quantification of DNA Transformation of plasmid into the HT115(DE3) cells	The day after lab: 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
9	11/4 - 11/10	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 "mock" plate for each genotype as well (6 plates total) Incubate at 23°C until next class
10	11/11 - 11/17	Examine the phenotypes of the fed worms - compare to control N2 worms and worms containing a mutation in the gene you are examining Collection of treated worms and RNA purification
11	11/29 - 12/3	Reverse transcription and PCR reactions Instructors run gels	Review and interpret the resulting gel images