Difference between revisions of "BISC219/F12:RNA interference"

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(New page: {{Template:BISC219/F12}} <div style="padding: 10px; width: 720px; border: 5px solid #7E354D;"> These labs were developed with the help of the '''Silencing Genomes''' project of the Dolan ...)
 
(Schedule of Experiments)
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! Lab # !! Dates !! Activity !! Outside lab time
 
! Lab # !! Dates !! Activity !! Outside lab time
|-
 
! 5
 
| 9/29 - 10/5
 
| Pick a gene of interest<br>
 
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<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
 
!7
 
|10/19 - 10/25
 
|10/19 - 10/25
|Colony PCR to check for transformation
+
|
 
|'''The night before next lab:'''<br>
 
|'''The night before next lab:'''<br>
Set up an overnight culture of a single colony from your transformation
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|-
 
|-
 
!8
 
!8
 
|11/2 - 11/8
 
|11/2 - 11/8
|Plasmid isolation from the BL21 cells <br>
+
|
Quantification of DNA<br>
 
Transformation of plasmid into the HT115(DE3) cells
 
 
|'''The day after lab:'''<br>
 
|'''The day after lab:'''<br>
Check control and transformation plates for growth - save your transformation plate<br>
 
 
'''The night before next lab:'''<br>
 
'''The night before next lab:'''<br>
Set up an overnight culture of a single colony from your transformation
 
 
|-
 
|-
 
!9
 
!9
 
|11/9 - 11/15
 
|11/9 - 11/15
|Induction of the bacteria to produce RNA<br>
+
|
Seed plates and dry for bacterial feeding RNAi<br>
 
 
|'''4 days later:'''<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
 
 
|-
 
|-
 
!10
 
!10
 
|11/16 - 11/22
 
|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<br>
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|  
Collection of treated worms and RNA purification
 
 
|
 
|
 
|-
 
|-
 
!11
 
!11
 
|11/28 - 12/2
 
|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.
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|-
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!12
 +
|12/ - 12/
 +
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Revision as of 12:19, 20 August 2012

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
7 10/19 - 10/25 The night before next lab:
8 11/2 - 11/8 The day after lab:

The night before next lab:

9 11/9 - 11/15 4 days later:
10 11/16 - 11/22
11 11/28 - 12/2
12 12/ - 12/