BISC 219/2009: Mod 1 Lab 2

Lab 2 Part 1: Scoring of the F2 Progeny from the Autosomal vs. X-linked Crosses

1. Examine each plate of F2 progeny. If you chose only L4 hermaphrodites you should only see hermaphrodite progeny. If you have a lot of males on your plates you chose young adult worms rather than L4's - see your instructor.
   
2. For each cross you will count and examine 100 worms - make this a random sample - the mutant worms may be smaller and not move as well as the wild types. Look around your plate.
   
3. Record in your lab notebook the number of WT, and Dpy, Unc or Dpy Unc mutants by examining the phenotype as you remove each animal from the plate (flame the pick to remove the worm). If unlinked, you should see WT's (+/+;+/+), Dpy’s (d/d;+/+), Unc’s(+/+;u/u) and Dpy Unc’s (d/d;u/u) in a ratio of 9:3:3:1. If linked, you should see a greater proportion of Dpy Unc’s (d u/d u) double mutants vs Dpy or Unc single mutants among the mutant hermaphrodite progeny.
   

You should now be able to conclude which strain is autosomal and linked, autosomal and unlinked and finally which strain has an x-linked gene and which one it is Dpy or Unc.

Lab 2 Part 2: Linkage Testing

Linkage Testing: you will determine on which of the five autosomes (linkage groups) your mutation is located. This task is a prerequisite to mapping. It is accomplished by determining the segregation behavior of your unmapped mutation relative to standard reference markers (e.g., mutations whose location is already known). Recall that unlinked mutations will segregate independently (your basic dihybrid inheritance as first observed by Gregor Mendel) whereas linked mutations will not.

In practice, linkage tests are performed using the following steps (where "d" (dpy) represents your recessive mutant tested with reference marker "u" (unc)). The markers d and u must be distinguishable. Since homozygous mutant males usually will not mate, the desired double heterozygote is constructed by mating males heterozygous for your dpy mutation but wild type for all other genes including the reference mutation (d/+;+/+) with hermaphrodites homozygous for the reference mutation unc(+/+; u/u). The genotypes of the F1 hybrids will be (+/d;u/+) and (+/+;u/+). We are only interested in the double heterozygote (+/d;u/+). The F1 hybrids containing only u are not useful. To select the (+/d;u/+) heterozygotes, we let 4 to 5 individual F1's self fertilize on their own individual plates (one on each plate). We score the progeny of the F1 individuals (the F2) for linkage. Only F1 worms which produce d/d homozygotes are scored, since those are the (+/d;u/+). This should be 50% of the plates.

F2 progeny of each class are counted in the (+/d;u/+) plates: wild-type (+/+;+/+); d (d/d;+/+); u (+/+;u/u) and du double (d/d;u/u). If assortment is independent, progeny will be: 9/16 wild type; 3/16 d, 3/16 u; 1/16 du (that is the 9:3:3:1 ratio)!

On the other hand, if the markers are closely linked double homozygotes (d u/d u) would occur only through a very rare recombination event, and thus you are not likely to observe the double mutant class.

To Do Today

1. For linkage testing set up four different crosses. Each cross will contain 3 heterozygous males (d/+) from the cross you initiated using your mutant Dpy worms. Make sure that these are the only animals that you transfer from that plate by transferring the males to a transfer plate and letting them crawl around for a minute - away from any contaminating worms - then pick a second time to the mating plate.
2. Each heterozygous (d/+) male will be mated to three L4 hermaphrodites that are homozygous for one of 4 known Unc (u/u) mutations on a mating plate. The strains and their reference mutations are: CB51 (unc-13 (I)); CB444 (unc-52 (II)); CB251 (unc-36 (III)); CB933 (unc-17 (IV)).
   
3. Label your four plates with your PURPLE Sharpie. With the genotype of the strain - for example: unc-13/unc-13; +/+ (H) X +/+; d/+ (M) with your initials and date.
4. Incubate all of the worms at 23°C for 3 days in your team's worm box.
   

3-4 days after lab:

1. For linkage testing, transfer 2 wild type cross progeny that are L4 stage (heterozygous for both traits) hermaphrodites from each of your 4 crosses to each of 2 new plates per cross for a total of 8 plates.
2. Label your 8 (4 sets of duplicates) plates with your PURPLE Sharpie. Label each plate with your initials, the genotype of your worms and the date. In each case, why is it important that you transfer L4’s and not adults? What is the genotype and phenotype of your expected F2 progeny?
   
3. Incubate all worms at 23°C until the next lab period.

Lab 2 Part 3: Calibration of Micropipettes

In this calibration you will measure the absorbance of a blue solution (0.01% BromoPhenolBlue) at various dilutions. The graph of absorbance versus volume of 0.01% BPB should be a straight line.

1. Transfer some deionized water from the tap to a 50 ml conical tube. Using your P1000, measure 1 ml of water into each of two 1.5ml plastic cuvettes. They will serve as auto zero blanks for the spectrophotometer.
2. Using your P20, measure 10, 15 and 20 microliters of 0.01% BPB solution into the bottom of three more cuvettes. Using your P1000, add water to bring the final volume to 1 ml in each cuvette.
3. Using your P200, measure 40, 100 and 200 microliters of 0.01% BPB solution into the bottom of three more cuvettes. Using your P1000, add water to bring the final volumes to 1 ml.
4. Using your P1000, measure 201, 300, and 400 microliters of 0.01% BPB solution into the bottom of three cuvettes. Add water to bring the final volume in each cuvette to 1 ml.
5. With a gloved hand or with a piece of parafilm over the lip of the cuvette, invert each cuvette several times to thoroughly mix the contents.
6. Measure the absorbance of each solution at 600 nm using one of the Hitachi double beam spectrophotometers (requiring two blanks) found in the equipment room, L308.
7. Open Excel on one of the computers in the back of the 219 lab and make a scatter plot of absorbance (plotted on the y-axis) versus volume of 0.01% BPB (plotted on the x-axis) for EACH pipette (in 3 separate graphs). The points should fall close to a straight line on each graph. Check the R2 values for the linear regression line on each graph. Each should be greater than 98%. If a linear regression line does not seem adequately linear, repeat of the calibration for that pipet and inform your instructor. It is possible that the micropipette needs cleaning but, more likely, that your technique needs adjusting. Send your excel spreadsheet to yourself via First Class. Tape a copy of your graphs into your lab notebook and also include the information from Table 1.

Clean up: The dilute solution of BPB can be discarded down the sink, the plastic tips can be discarded into the autoclave bags above your bench, and the plastic cuvettes, paper towels, etc into the regular trash cans. Please do NOT place materials (other than pipet tips) that are not contaminated with microorganisms into the autoclave bags.

Note: Another way to calibrate your P1000 is to measure the weight of 1000 microliters of water. This volume should weigh 1 gram at room temperature.

Table 1 Calibration of Micropipettes

Micropipette	volume pipetted	volume of water added	OD600
P20	10 μl
	15 μl
	20 μl
P200	40 μl
	100 μl
	200 μl
P1000	201 μl
	300 μl
	400 μl

HITACHI SPECTROPHOTOMETER OPERATION
TO READ ABSORBANCE (the machine requires at least 10 min. to warm up after turning it on before use):
MAIN MENU KEY
Press ENTER (2X)
Under Data Mode, select 1 for absorbance, ENTER
Test Set Up, ENTER
Sample num., select 1, ENTER
Wavelength, set desired wavelength, ENTER
Delay, set 1 or 2 seconds, ENTER
FORWARD KEY (wait for proper wavelength to appear on screen)
Place blank material in the reference and test positions, AUTO ZERO KEY
Remove blank material from test position and replace with sample, START KEY
Repeat for remaining samples, PAPER FEED KEY, take printed results and record in your lab notebook.

C. elegans General Information
Tools and Techniques
Lab 1: Welcome to C. elegans and Mutant Hunt
Lab 3: Linkage Test, Backcross and Mapping
Lab 4: Mapping Part 2
Lab 5: Score!