BISC 219/F10: RNAi Lab 5
Lab 5: Series 3-Reverse Genetic Analysis: Picking a Gene
In the age of genome sequencing we now know or can make educated guesses about the location of every gene in an organisms genome. This does not give us any information about the function of the gene product (protein) in the organism though. We can use reverse genetics to help us solve this puzzle. There are several tools in the reverse genetics toolbox: directed mutation (point mutations or deletions), overexpression using transgenes, and gene silencing using double stranded RNA (RNAi). Only the latter two have been perfected in C. elegans. Scientists still have not found a way to do in vivo homologous recombination in worms.
We are going to use RNAi as our tool to investigate gene function via reverse genetics. C. elegans is the first animal in which the process of RNAi was discovered. A similar system was identified in plants years earlier but sadly was largely ignored by the scientific community. We now know that RNA regulation in cells is a fundamental method of regulating gene expression in organisms from microscopic C. elegans to humans. Many labs are now working non-stop to develop treatments for many "incurable" diseases using RNAi.
You will have available to you DNA from 2-3 genes of interest. Each pair will clone a piece of one gene from a non-RNAi plasmid into a plasmid that will allow us to produce double stranded RNA inside bacteria. These bacteria will then serve as food for our C. elegans and induce the RNAi pathway in the worms, knocking down the amount of mRNA specific to that gene inside the cell and thus the amount of protein in the cells and possibly inducing a phenotype.
Calibration of Micropipettes
- To calibrate your P1000, P 200, and P 20 micropipets, label 6 microfuge tubes (1-6) and weigh them. Record the weights in the table below.
- Following the table below, pipet the specified volumes into the pre-weighed microfuge tubes prepared above and then re-weigh them. Record all weights.
- Calculate the weight of the water in grams. 1000 microliters of water should weigh 1 gram at room temperature.
- If the water in any tube weighs more or less than 1 gram, ask your instructor for help. If your calibration is significantly off after several repeated attempts, your pipet (or your technique!) may need adjustment.
| Tube # | Tube Pre-Weight | Vol. in μL using P20 | Vol. in μL using P200 | Vol. in μL using P1000 | Weight of Tube + Water in grams | Weight of Water in grams |
|---|---|---|---|---|---|---|
For a Word™ format protocol: Media:Protocol for Micropipet Calibration.doc
C. elegans PCR
Each master mix includes:
45 μL of PCR buffer (10 mM Tris, 50 mM KCl, 1.5 mM MgCl2 pH 8.3)
1 μL of 10 mM dNTPs
1 μL of forward primer (20 μM stock)
1 μL of reverse primer (20 μM stock)
1 μL of 5 units/μL Taq Polymerase
1 ul of "library DNA"
Total PCR reaction volume = 50 μL
Primer sequences:
| Gene name | Forward Primer | Reverse Primer |
|---|---|---|
| rol-5 | 5' ATG CAT AGA TCT GAT TAT CTG CTC CAC CAG GTC AAC CAC C 3' | 5' ATG CAT AAG CTT TTA GAT ATT TCT GTA TCC ACG G 3' |
| lon-2 | 5' ATG CAT AAG CTT GAT TTA GGC GAT TTC CTT GAC TGG AAT C 3' | 5' ATG CAT AGA TCT TTT GCA CAC ACC AAG ACC ATG CAC TCC C 3' |
| vab-10 | 5' ATG CAT ACT AGT ATG ATG GAG AAT CGG AAG AAA CCA TCT 3' | 5' AGA TCT GAT TTT GTT CAT GGC AAT CGT GTT GGT C 3' |
| bli-1 | 5' ATG CAT AGA TCT CAC ACC GAC AAA CTC CAC ACG AGT TGT A 3' | 5' ATG CAT ACT AGT TTA TTA TGG CAA GTG GGG GAA GGG GTG A 3' |
PCR Conditions:
| Step | Temperature | Time | Repeat |
|---|---|---|---|
| 1 | 94°C | 2 minutes | 1 time |
| 2 | 94°C | 30 seconds | |
| 3 | 54°C | 30 seconds | |
| 4 | 72°C | 2 minutes | Steps 2-4 30 times total |
| 5 | 72°C | 10 minutes | 1 time |
| 6 | 4°C | forever | end program |
Agarose gel electrophoresis of PCR product:
(NOTE: Because of the length of time it takes to run the PCR reactions - agarose gels will be run by the instructors.)
Instructors will prepare a sample for electrophoresis by mixing 10 μL of your PCR product with 1-2 μL of loading dye and then add it to a single well on a 1.0% agarose gel with SybrSafe™ stain. Bands will be separatedRun the gel at 100V for 45 minutes to an hour. The gel will be photographed using UV light and the photo posted to the lab conference.
The expected DNA fragment sizes are:
lon-2:1586 bp
rol-5:718 bp
vab-10:1172 bp
bli-1:1070 bp
Assignment
Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab.
Links to Labs& Project Info
Series1:
Worm Info
Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start
Lab 2: Sex-Linked or Autosomal Finale
Series2:
Background: Classical Forward Genetics and Gene Mapping
Lab 2: Mutant Hunt
Lab 3: Linkage Test Part 1
Lab 4: Linkage Test Part 2, Mapping and Complementation
Lab 5: Finish Complementation; Mapping Con't
Lab 6: DNA sequence analysis; Mapping Con't
Lab 7: Complete Mapping: Score
Series3:
Schedule of Reverse Genetics Project
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
