BISC 219/2009:RNA interference

From OpenWetWare
Jump to navigationJump to search
Wellesley College BISC 219 Genetics

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