BISC 219/F10: RNAi Lab 5

Lab 5: Picking a Gene for Reverse Genetic Analysis

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.

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' 3'	5' 3'
lon-3	5' 3'	5' 3'
vab-10	5' 3'	5' 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-3:
rol-5:
vab-10:

RNAi General Information
Media Recipes
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