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 ...)
Current revision (21:04, 18 November 2012) (view source)
 
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<div style="padding: 10px; width: 720px; border: 5px solid #7E354D;">
<div style="padding: 10px; width: 720px; border: 5px solid #7E354D;">
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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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These labs were modified from the work of Janet Andersen, Alexander Krichevsky, Joerg R. Leheste, and Daniel J. Moloney  at the Dept. of Biochemistry and Cellular BIology at Stony Brook University with the '''Silencing Genomes''' project of the Dolan DNA Learning Center in Cold Spring Harbour, New York. providing some of the worm strains.<br><br>
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[[BISC219/F12: RNAi General Information| RNAi General Information]] <br>
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[[BISC219/F12: RNAi General Information| Background Information on Project 3: Investigating Gene Function & Regulation Using RNAi]] <br>
[[BISC219/F12: Media Recipes | Media Recipes]]<br>
[[BISC219/F12: Media Recipes | Media Recipes]]<br>
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[[BISC219/F12: RNAi Lab 5 | Lab 5: Picking your gene to RNAi]]<br>
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[[BISC219/F12: RNAi Lab 7 | Lab 7: Identifying a bacterial colony containing our plasmid of interest  ]]<br>
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[[BISC219/F12: RNAi Lab 6 | Lab 6: Cloning your gene of interest]]<br>
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[[BISC219/F12: RNAi Lab 8 | Lab 8: Creating the feeding strain of bacteria for RNAi]]<br>
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[[BISC219/F12: RNAi Lab 7  | Lab 7: Picking your transformant]]<br>
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[[BISC219/F12: RNAi Lab 9 | Lab 9: Induction of feeding strain to produce dsRNA and feeding worms]]<br>
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[[BISC219/F12: RNAi Lab 8 | Lab 8: Plasmid purification and transformation]]<br>
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[[BISC219/F12: RNAi Lab 10 | Lab 10: Phenotypic analysis of treated vs untreated worms]]<br>
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[[BISC219/F12: RNAi Lab | Lab 9: Induction of bacteria for RNAi]]<br>
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[[BISC219/F12: RNAi Lab 11 | Lab 11: Data Analysis Workshop]]<br>
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[[BISC219/F12: RNAi Lab 10 | Lab 10: Scoring your worms ]]<br>
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[[BISC219/F12: RNAi Lab 12 | Lab 12: Optional Conferences]]<br>
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[[BISC219/F12: RNAi Lab 11 | Lab 11: Individual Writing Conferences]]<br><br>
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! Lab # !! Dates !! Activity !! Outside lab time
! Lab # !! Dates !! Activity !! Outside lab time
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|-
 
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! 5
 
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| 9/29 - 10/5
 
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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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|10/12 - 10/18
 
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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>
 
|-
|-
!7
!7
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|10/19 - 10/25
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|10/23 - 10/29
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|Colony PCR to check for transformation
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| Picking a colony of bacteria containing the RNAi plasmid<br>
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|'''The night before next lab:'''<br>
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PCR to confirm presence of gene of interest
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Set up an overnight culture of a single colony from your transformation
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|'''The night before next lab:''' set up an overnight culture of your colony of interest<br>
|-
|-
!8
!8
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|11/2 - 11/8
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|10/30 - 11/5
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|Plasmid isolation from the BL21 cells <br>
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| Miniprep of plasmid and transformation into feeding bacteria strain
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Quantification of DNA<br>
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|'''The day after lab:''' check your transformed bacteria for colony growth<br>
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Transformation of plasmid into the HT115(DE3) cells
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'''The night before next lab:''' set up an overnight culture of your colony of interest<br>
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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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'''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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|-
|-
!9
!9
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|11/9 - 11/15
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|11/7 - 11/13
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|Induction of the bacteria to produce RNA<br>
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| Induction of dsRNA production, seeding of RNAi plates and addition of worms
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Seed plates and dry for bacterial feeding RNAi<br>
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|
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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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|-
|-
!10
!10
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|11/16 - 11/22
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|11/14 - 11/20
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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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| Chemotaxis Assay
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Collection of treated worms and RNA purification
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|
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!11
!11
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|11/28 - 12/2
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|11/26 - 11/30
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|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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| Writing Workshop
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|-
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!12
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|12/3 - 12/7
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| Writing Conferences
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|  
|-
|-

Current revision

Wellesley College BISC 219 Genetics

These labs were modified from the work of Janet Andersen, Alexander Krichevsky, Joerg R. Leheste, and Daniel J. Moloney at the Dept. of Biochemistry and Cellular BIology at Stony Brook University with the Silencing Genomes project of the Dolan DNA Learning Center in Cold Spring Harbour, New York. providing some of the worm strains.

Background Information on Project 3: Investigating Gene Function & Regulation Using RNAi
Media Recipes
Lab 7: Identifying a bacterial colony containing our plasmid of interest
Lab 8: Creating the feeding strain of bacteria for RNAi
Lab 9: Induction of feeding strain to produce dsRNA and feeding worms
Lab 10: Phenotypic analysis of treated vs untreated worms
Lab 11: Data Analysis Workshop
Lab 12: Optional Conferences


Schedule of Experiments

Lab # Dates Activity Outside lab time
7 10/23 - 10/29 Picking a colony of bacteria containing the RNAi plasmid

PCR to confirm presence of gene of interest

The night before next lab: set up an overnight culture of your colony of interest
8 10/30 - 11/5 Miniprep of plasmid and transformation into feeding bacteria strain The day after lab: check your transformed bacteria for colony growth

The night before next lab: set up an overnight culture of your colony of interest

9 11/7 - 11/13 Induction of dsRNA production, seeding of RNAi plates and addition of worms
10 11/14 - 11/20 Chemotaxis Assay
11 11/26 - 11/30 Writing Workshop
12 12/3 - 12/7 Writing Conferences