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

Latest revision as of 09:51, 14 August 2010

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