BISC 219/2009:RNA interference

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Wellesley College BISC 219 Genetics

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Gene Mapping        RNA interference        Creating a Transgenic Organism        Assignments       

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