BISC 219/F10: RNAi Lab 5

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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 feeder 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 at | http://www.dnalc.org/resources/animations/pcr.html

(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 page at | http://www.dnalc.org/resources/animations/)

PCR Master Mix contains:
      40 ul H2O
      5 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

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 micropipet 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 pipet 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 at | http://www.dnalc.org/resources/animations/gelelectrophoresis.html .

(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 New England BIolabs (ladder #323-1S 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

Outline of Experimental Design for REVERSE Genetics Project

Where are you now in this process?(What have you done so far; What's next?)
Make the feeder strain of bacteria

  1. Amplify gene of interest by pcr ;
  2. Restriction Enzyme digestion of amplified DNA to create "sticky ends" for ligation;
  3. Clean up DNA (remove enzymes);
  4. Cloning: ligate gene into vector plasmid with amp resistance gene ;
  5. Transform competent bacterial cells;
  6. Select for transformants on media with ampicillin;
  7. Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interst
  8. Culture the selected colony from colony pcr to create a lot of copies of these bacteria
  9. Isolate the cloned plasmid DNA from that cultured colony by miniprep;
  10. Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA;
  11. Select for transformants on media with ampicillin
  12. Choose an isolated colony to culture and make lots of feeder strain bacteria;
  13. Induce expression of C. elegans gene dsRNA from the pL4440 vector in the bacteria by IPTG induction.
  14. Seed NM lite worm growth media plates with feeder strain produced as described

Plate wild type C. elegans worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest).

Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.

Isolate RNA from RNAi worms and control worms of same strains.

Perform RT-PCR (Reverse Transcriptase)to amplify the C. elegans gene of interest, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.

Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare the amount of amplified gene of interest in RNAi treated vs. untreated worms.

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.

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