Difference between revisions of "BISC219/F11:RNA interference"

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Latest revision as of 09:02, 8 November 2011

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
Lab 11: Individual Writing Conferences


Schedule of Experiments

Lab # Dates Activity Outside lab time
5 9/29 - 10/5 Pick a gene of interest

Set up a PCR reaction to clone the gene

Examine the results of the agarose gel electrophoresis
6 10/12 - 10/18 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 10/19 - 10/25 Colony PCR to check for transformation The night before next lab:

Set up an overnight culture of a single colony from your transformation

8 11/2 - 11/8 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 11/9 - 11/15 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 11/16 - 11/22 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 11/28 - 12/2 Sign up for a time to meet and discuss your final paper draft with your instructor - we will not meet in lab this week.