BISC219/F11:RNA interference

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(New page: {{Template:BISC219/F11}} <div style="padding: 10px; width: 720px; border: 5px solid #6600CC;"> These labs were developed with the help of the '''Silencing Genomes''' project of the Dolan ...)
Current revision (12:02, 8 November 2011) (view source)
 
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Current revision

Wellesley College BISC 219 Genetics

Home     People/Info     Lab Calendars/Weekly Planner     OWW Basics     Resources       Glossary    
Series1:Sex-linked or Autosomal        Series2:Classical (Forward) Genetics        Series3: Reverse Genetics Using RNAi
       Worm Info     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 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.