Lab 7: Series2 Forward Genetics Project- SCORE!
Mapping: If you haven't already done so, form a hypothesis about your expected recombination frequency (RF) for the test cross you will score today. RF is directly proportional to map units (in centimorgans cM) a measurement of the distance that linked genes are apart. You can check previous research (Wormbase is a good place to look) for the number of map units between the your linkage group (gene of interest and reference gene). How many recombinants (total) do you expect in each 100 worms scored? Note that it is difficult to translate map distance in cM to distance between genes in bases. The conversion factor varies among species and changes as distance between genes changes. Therefore, we will limit our mapping to calculating distances in map units.
Scoring the Test Cross: Each partner should count all the adult progeny from one test cross plate, scoring the phenotype as either wild type , Dpy , Unc or Dpy Unc. Remember to remove each animal after you have determined its phenotype. Flame the WT and UncDpy progeny but transfer any single mutants to a new plate. When you have finished scoring your plate, ask both your partner and your lab instructor to confirm that the worm progeny that you scored as either Dpy or Unc are,in fact, single mutants. Record your totals on the google spreadsheet provided for course data. This spreadsheet will update as each lab section enters its data. You can find this spreadsheet in the DATA file in Resources in your lab section's Sakai site and on a computer in the back of the lab. You can calculate RF and map distance from your own group's and class' data before we have the course data completed, but, of course, the evidence used in your paper will be the RF calculated from the full data set.
Review the crosses that you diagrammed for this mapping project and make sure you understand how the test cross we set up differentiates, phenotypically, progeny of parental gametes from the progeny of recombinant gametes. Hint: how did you end up with either of the single mutant classes ( Unc or Dpy) from these parental genotypes d u/+ + (genotype of the male) and d u/ d u (genotype of the hermaphrodite parent)?
You will determine map distance of the dpy gene of interest from an unc gene using the formula: RF (recombinant frequency) =the number of single mutants (both dpy and unc single mutants) divided by the total number of worms counted * 100 (to obtain RF in % recombinants and thus in map units).
Congratulations! You now have calculated the location of the dpy mutation in map unit distance away from your reference linked unc gene on the a particular autosome. To check your location (and the accuracy of your recombination frequency relationship to map units), enter the linked unc gene name into Wormbase. Scroll down and click on Location and Mapping Data and find the gene that is your calculated number of map units away from the linked unc gene. (Remember that you will have to look in both directions on the chromosome). Is the dpy gene that you found allelic in complementation analysis at one of these map locations? Yes? Terrific! If not, your next step would be to see what is known about the gene or ORF at those locations on the chromosome and see if what's known fits in at all with your observations. If it does, great and, if not, you have some thinking to do about the identity of your dpy gene of interest. (You will NOT write a paper about "sources of error" in your experimental design or your execution of the experiment!!!!)
If you think you know the identity of your dpy gene, enter this name into the box at the top of the page in Wormbase and click Search. It will either bring you directly to that page or it will bring you to a page with multiple hits - click on the link that provides a definition for what the gene does.
On this new page should be all the known information about this particular gene. Its name, who named it, what the gene encodes - if that is known, and much more. At the bottom will be a list of references - or a link to a list of references. Find out the function of this gene.
Spend some time with Wormbase and marvel at all the hard work and years of research that went into discovering all this information about this tiny little soil nematode that causes us no harm (non-parasitic). Why do you think so many smart people have devoted so much of their time and energy to working out the genetics of "appearance or movement challenged" little worms? We will talk more about model organisms and the power of functional and comparative genomics in our next series.
Lab 7: Series 3 - Examining the effect of heat shock on the CL2070 strain
Instructors will do before lab:
Your instructor or our lab specialist will come in 4-5 hours before lab and "heat shock" one plate each of your worms that have been incubating at 15°C and 23°C. The second plate at each temperature serves as your control. Heat shocking involves moving the worms to a 37°C incubator for 1 hour. The worms will then be placed back at their proper temperature until you use them in lab.
To Do in Lab Today
We only have one fluorescent dissecting scope for viewing the worms. While some groups are scoring their worms others will be working with the instructor in the microscope room to view and photograph their worms.
- Bring your 4 plates of worms down to the microscope room, L318C.
- You will look at your worms and take some pictures under the dissecting scope. Follow the directions below for visualizing and photographing your worms. Are the worms glowing? What part of the worms are glowing?
Directions for Using the Leica M165 Dissecting Fluorescence Microscope in L318C
Directions for Using the Leica M165 Dissecting Fluorescence Microscope in L318C
Control Box: power button (green).
Microscope: Power switch (right rear of the microscope)
Camera/Microscope: On/Off switch in the middle of the rear tower of the microscope
Computer software for the camera: If the computer is not already on, turn on CPU for the PC to the right of the microscope and log in. Open Leica camera software LASV3.8 by double clicking on icon on the Desktop. (Ignore the message that says license is expired if you get that). This step may take awhile.
Before viewing fluorescence, get specimen into focus using white transmitted light.
Adjustments for white light:
a. Front and rear small knobs on right side of microscope adjusts shutter and light intensity.
b .Coarse and fine focus controlled by large and smaller black knobs on both sides of microscope.
c. Magnification controlled by large, white knob on both sides of microscope anterior to focus control.
d. You can adjust contrast/phase using the front slide knob on the left side of the microscope.
e. Make sure small knob on left side of microscope is set to VIS (not DOC!)
For GFP excitation:
1. Turn on shutter control (yellow light) on Power Box (near green on/off switch) when ready to view fluorescence.
2. Make sure large round black wheel in middle of microscope is set to the right excitation filter. For GFP it is GFP2.
3. Turn white light off using rear shutter control knob described in a. Turn it fully to the rear.
4. Open shutter for fluorescence by turning wheel on right side of microscope (connected to the wire to the Control box) to the open circle position.
5. Make sure small knob on left side of microscope is set to VIS (not DOC)
6. Use focus and field adjustment knobs to find your specimen and focus it.
To Take A Photo:
1. Change position of small knob described in 5. To DOC (not VIS).
2. On the MIC1 menu on the computer screen Make sure GFP2 ET is highlighted (if you are looking at GFP).
3. Use the Exposure and Gain sliders on the computer screen to adjust the exposure and contrast to view your image.
4. Adjust the fine focus on the microscope while looking at the image on the computer screen.
5. When you are ready to capture the image, click Acquire on the bottom of the computer screen.
6. A group of previous images should show up on the bottom of the computer screen in thumbnails. Yours should be the one on the far left. When you have found your image right click on it once. Click Export and change the settings from .tiff to .jpg and downgrade the Quality menu to HIGH Quality (smaller file!). Rename the file giving the strain name (CL2070), temp grown (15 or 23C), heat shock (HS) or not, and treatment: control, RNAi treated or untreated. Also include lab day (M,T, W, Th, or F) and team color .
7. Minimize the screen by clicking on – in the top right corner (DO NOT close the program by clicking X!!)
8. View the Desktop of the computer and find the folder with your Lab Instructor’s name (usually the top left). If you have logged in under your own name, a folder with your name should be on the desk top instead of your instructor’s. Open the folder and find and open the subfolder called PICTURES.
9. You should be able to find your images and email them to yourself AND/or upload them to a folder in our lab Sakai site found under Resources--DATA--Project 3---GPF images—Team COLOR
10. When your images are uploaded and saved in Sakai, maximize the LASV3. 8 software screen and return to the Mic1 menu tab (away from the Camera tab next to it).
11. Don’t turn off anything until the whole class has completed their observations and taken all of their photos.
12. The Last user (probably your lab instructor) will turn off shutter, microscope, camera and power source. Clean microscope lens with lens paper.
13. DO NOT TURN OFF COMPUTER BUT DO LOG OUT.
If the Computer Crashes, restart the CPU by pushing the On button and hit RETURN when the computer attempts to restart and asks you if it should do that.
To do on the day before Lab 8:
You and your partner will return to the lab to make an overnight broth culture of one of the colonies of E. coli bacteria containing a specially engineered plasmid called pPD129.36 hsf-1. The sub-culture of these bacteria that you will set up tonight will create many identical copies of cells that carry the plasmid containing your gene of interest.
- Find the LB+amp plate in the white refrigerator in the back of the lab on the left. This plate contains solid medium and bacterial colonies. The plate you should use is labeled with your lab day.
- Begin by obtaining two tubes of LB broth (each will have 5 ml of broth) from the same refrigerator.
- Add 5 microliters of the 50mg/ml ampicillin stock to each tube. The antibiotics can also be found in the refrigerator in a small box with microfuge tubes labeled with the name and concentration of the antibiotic. Calculate the effective concentration of ampicillin that you will have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook.
- Add 5 microliters of the 12.5mg/ml tetracycline stock to each tube. (Tetracycline stock is also found in microfuge tubes in the same box with the ampicillin stock). Calculate the effective concentration of tetracycline that you will have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook.
- Gently swirl your LB +amp + tet broth to mix the contents.
- Label the two sterile glass culture tubes with tape in your team color. Label one with "pPD129.36 hsf-1" and your initials. Label the other with your initials and Control.
- Inoculate the broth that is not your control with bacteria by using a sterile disposable loop to scrape (lightly!) a medium size, single, well isolated colony off the plate. Be sure not to touch the plate with the loop except on the desired colony to avoid picking up any satellite colonies! Gently swirl the loop in the LB+amp+tet broth - you should be able to see the colony come off the loop. (The control tube should not be inoculated with bacteria.)
- Locate the rotating wheel in the 37C incubator by the door to the lab.
- Balance the 2 tubes across from each other and turn the wheel on. (If you need to turn off the wheel briefly to position your tubes, do so.)
- Incubate these broth cultures at 37°C overnight. Do not forget to make sure the wheel is rotating when you leave!
Scientific Research Report on Series2 work Classical (Forward) Genetics is due at the beginning of Lab 9. Assignment information found at BISC219/F13: Assignment_ Series2_Classical Genetics Paper