BISC 219/F10:RNA interference: Difference between revisions
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[[BISC 219/F10:RNAi General Information| RNAi General Information]] <br> | [[BISC 219/F10:RNAi General Information| RNAi General Information]] <br> | ||
[[BISC 219/F10:Media Recipes | Media Recipes]]<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: ]]<br> | |||
[[BISC 219/F10: RNAi Lab 7 | Lab 7: ]]<br> | |||
[[BISC 219/F10: RNAi Lab 8 | Lab 8: ]]<br> | |||
[[BISC 219/F10: RNAi Lab 9 | Lab 9: ]]<br> | |||
[[BISC 219/F10: RNAi Lab 10 | Lab 10: ]]<br> | |||
[[BISC 219/F10: RNAi Lab 11 | Lab 11: ]]<br><br> | |||
=='''Schedule of Experiments'''== | |||
{| border="1" | |||
|+ | |||
! Lab # !! Dates !! Activity !! Outside lab time | |||
|- | |||
! 5 | |||
| 10/5 - 10/12 | |||
| Pick a gene of interest<br> | |||
Set up a PCR reaction to clone the gene | |||
|Examine the results of the agarose gel electrophoresis | |||
|- | |||
!6 | |||
|10/13 - 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 | |||
|- | |||
!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 | |||
|- | |||
!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<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 | |||
|- |
Revision as of 09:49, 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:
Lab 7:
Lab 8:
Lab 9:
Lab 10:
Lab 11:
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 |
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 |
The day after lab: Check control and transformation plates for growth - save your transformation plate |
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) |
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 |