BISC219/F12: RNAi Lab 7

Lab 7: Series 3- Investigating Gene Regulation Using RNAi:

In the age of genome sequencing we now know, or can make educated guesses about, the location of every gene in an organism's genome; however, this does not give us any information about the function of the gene product (protein) in the organism. We can use reverse genetic analysis 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 knockout organisms or double stranded RNA (RNAi). Only RNAi and overexpression 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 the lsy-2 gene. Each pair will obtain bacteria containing 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 the lsy-2 gene inside the cell and thus the amount of protein in the cells and possibly inducing a phenotype.

Picking a Bacterial Colony

Your instructor will give each pair an agar plate on which bacteria containing our pPD129.36 lsy-2 plasmid are selectively growing. Please pick a well isolated colony, circle it, write your initials and group color on the agar side of the plate and wrap it in Parafilm. Store the plate in the refrigerator unti the day before the next lab when you will set up an overnight culture.

Calibration of Micropipettes

1. 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.
2. Following the table below, pipet the specified volumes into the pre-weighed microfuge tubes prepared above and then re-weigh them. Record all weights.
3. Calculate the weight of the water in grams. 1000 microliters of water should weigh 1 gram at room temperature.
4. 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
1		10	0	990
2		0	100	900
3		20	175	805
4		2	88	910
5		0	200 (5 times)	0
6		20 (5 times)	0	900

For a Word™ format protocol: Media:Protocol for Micropipet Calibration.doc