BISC219/F11: 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 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 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

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

C. elegans PCR

In order to begin the process of making a feeding strain of bacteria that will express dsRNA of your gene of interest and initiate RNAi in the worms, we must first get a lot of copies of our gene. We will do this through a well known and often used molecular tool called a polymerase chain reaction (PCR) reaction. PCR accomplishes an exponential amplification of a DNA template, the specificity of which is determined by short sequences of dsDNA called "primers". To see an animation of this process go to this link to the DOLAN DNA center.

(FYI: Other animations of some of the other molecular tools we'll be using in our reverse genetics project can also be found on the Dolan site index.

PCR Master Mix contains:
      40 ul H₂O
      5 μL of 10X 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

You will need to add:
      1 ul of "library DNA" containing template for your gene of interest

Total PCR reaction volume = 50 μL

NOTE:Polymerase Chain reactions are highly prone to contamination so use your best technique when pipetting reagents for PCR. Wash your hands, wear gloves and DON'T touch the micropipette tips or the inside of the PCR tube top. Since 1μL is close to nothing and the proper ratio of template DNA is crucial in a PCR, you must BE VERY CAREFUL when you pipette the DNA. Look at your pipet tip after you have drawn up the measured volume and be sure there is liquid in the tip. Dispense it near but not in the Master Mix so you can see that you have dispensed a bead of liquid into the tube. Make sure you tap or pulse the pcr tube in an adapter in microcentrifuge to get rid of bubbles and to make sure all the ingredients are mixed and in the bottom of the tube where they can interact.

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:

Agarose Gel electrophoresis is one of the most commonly used lab tools in molecular biology for visualizing isolated or separated fragments of DNA. To see more about this tool, visit the DOLAN DNA center's animation.

(NOTE: Because of the length of time it takes to run the PCR reactions - agarose gels may be run by the instructors if we run out of time today.)
Prepare a sample for electrophoresis by removing 10 μL of your PCR product and placing it into a clean microfuge tube labeled with your team color. Add 1-2 μL of loading dye that can be found at the instructor's bench. The ingredients and concentration of the loading dye and the buffer used in this electrophoresis can be found in the Media Recipes page. Mix by "flicking" and add all 12μL to a single well on the prepared 1.0% agarose gel containing SybrSafe™ DNA stain (from Invitrogen: the stain is diluted 1:10,000 in buffer). Identify your sample by putting your team color on the appropriate well of the template provided.Your instructor will add the DNA ladders and turn on and off the current (run the gel for ~ 45 min. at 100 V) and photograph the gel under UV light. She will post the gel photo to the data folder on the course conference so you can analyze it for successful amplification of your gene of interest. Our DNA standards ladder is from NEB Ladder N323-1S (used at 500μL/ml).

DNA fragments, seen as "bands" will be separated by molecular weight when you subject the the DNA to electrophoresis (current running from negative to positive electrodes). The negatively charged DNA will migrate through the fibers of the agarose toward the positive pole but at different speeds. Smaller DNA fragments (in basepairs) will make their way through the gel faster than larger fragments. A comparative set of DNA fragments of known size (called a ladder) will be run for comparison with the amplified DNA in your PCR product so that you can determine the size and purity of your amplified DNA and the success of the amplification. The separated DNA on the gel is stained with a non-toxic fluorescent DNA binding agent and can be photographed using UV light.

The expected DNA fragment sizes are:
lon-2:1586 bp
rol-5:718 bp
vab-10:1172 bp
bli-1:1070 bp