BISC 219/F10:RNA interference

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These labs were developed with the help of the '''Silencing Genomes''' project of the Dolan DNA Learning Center.<br><br>
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[[BISC 219/F10:RNAi General Information| RNAi General Information]] <br>
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[[BISC 219/F10:Media Recipes | Media Recipes]]<br>
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[[BISC 219/F10: RNAi Lab 5  | Lab 5: Picking your gene to RNAi]]<br>
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[[BISC 219/F10: RNAi Lab 6  | Lab 6: Cloning your gene of interest]]<br>
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[[BISC 219/F10: RNAi Lab 7  | Lab 7: Picking your transformant]]<br>
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[[BISC 219/F10: RNAi Lab 8  | Lab 8: Plasmid purification and transformation]]<br>
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[[BISC 219/F10: RNAi Lab 9  | Lab 9: Induction of bacteria for RNAi]]<br>
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[[BISC 219/F10: RNAi Lab 10 | Lab 10: Scoring your worms and RNA purification]]<br>
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[[BISC 219/F10: RNAi Lab 11 | Lab 11: RT PCR reactions]]<br><br>
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=='''Schedule of Experiments'''==
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{| border="1"
 +
|+
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! Lab # !! Dates !! Activity !! Outside lab time
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|-
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! 5
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| 10/5 - 10/12
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| Pick a gene of interest<br>
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Set up a PCR reaction to clone the gene
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|Examine the results of the agarose gel electrophoresis
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|-
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!6
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|10/13 - 10/19
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|Restriction enzyme digest of PCR product<br>
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Cleanup and ligation into the pPD129.36 vector<br>
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Transformation of the isolated plasmid into the BL21 cloning ''E. coli''
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|'''The day after lab:'''<br>
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Check control and transformation plates for growth - save your transformation plate<br>
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|-
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!7
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|10/20 - 10/26
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|Colony PCR to check for transformation
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|'''The night before next lab:'''<br>
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Set up an overnight culture of a single colony from your transformation
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|-
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!8
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|10/28 - 11/3
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|Plasmid isolation from the BL21 cells <br>
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Quantification of DNA<br>
 +
Transformation of plasmid into the HT115(DE3) cells
 +
|'''The day after lab:'''<br>
 +
Check control and transformation plates for growth - save your transformation plate<br>
 +
'''The night before next lab:'''<br>
 +
Set up an overnight culture of a single colony from your transformation
 +
|-
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!9
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|11/4 - 11/10
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|Induction of the bacteria to produce RNA<br>
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Seed plates and dry for bacterial feeding RNAi<br>
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|'''4 days later:'''<br>
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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''')<br>
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Incubate at 23°C until next class
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|-
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!10
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|11/11 - 11/17
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| Examine the phenotypes of the fed worms - compare to control N2 worms and worms containing a mutation in the gene you are examining<br>
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Collection of treated worms and RNA purification
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|
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|-
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!11
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|11/29 - 12/3
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| Reverse transcription and PCR reactions<br>
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Instructors run gels
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| Review and interpret the resulting gel images
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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 and RNA purification
Lab 11: RT PCR reactions


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
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