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
{| border="1" ! Lab # !! Dates !! Activity !! Outside lab time ! 5 Set up a PCR reaction to clone the gene !6 Cleanup and ligation into the pPD129.36 vector Transformation of the isolated plasmid into the BL21 cloning E. coli Check control and transformation plates for growth - save your transformation plate !7 Set up an overnight culture of a single colony from your transformation !8 Quantification of DNA Transformation of plasmid into the HT115(DE3) cells 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 Seed plates and dry for bacterial feeding RNAi 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 Collection of treated worms and RNA purification !11 Instructors run gels
 * 10/5 - 10/12
 * Pick a gene of interest
 * Examine the results of the agarose gel electrophoresis
 * 10/13 - 10/19
 * Restriction enzyme digest of PCR product
 * The day after lab:
 * 10/20 - 10/26
 * Colony PCR to check for transformation
 * The night before next lab:
 * 10/28 - 11/3
 * Plasmid isolation from the BL21 cells
 * The day after lab:
 * 11/4 - 11/10
 * Induction of the bacteria to produce RNA
 * 4 days later:
 * 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
 * 11/29 - 12/3
 * Reverse transcription and PCR reactions
 * Review and interpret the resulting gel images