BISC 219/F10:RNA interference

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Wellesley College BISC 219 Genetics

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Series1:Sex-linked or Autosomal        Series2:Classical (Forward) Genetics        Series3: Reverse Genetics Using RNAi
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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:
Lab 7:
Lab 8:
Lab 9:
Lab 10:
Lab 11:


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