BISC 219/2009: Mod 1 Lab 2

Lab 2 Part 1: Scoring Autosomal vs. X-linked

Record in your lab notebook the number of WT, Dpy, Unc and Dpy Unc mutants by scoring the phenotype and removing that animal from the plate (flame the pick to remove the worm). If unlinked, you should see WT's, Dpy’s, Unc’s and Dpy Unc’s in a ratio of 9:3:3:1. If linked, you should see greater than 1/7th Dpy Unc’s among the mutant 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 and Backcross

Once you have recovered your mutant and confirmed its phenotype (by examining its progeny) you will next perform two tasks in parallel. One of these is to backcross the mutation into wild type. This consists of crossing your mutant times wild type and recovering homozygous mutants in the F2 generation. Backcrossing is necessary to eliminate any secondary mutations that might modify the phenotypes you are studying. This is important because most mutant hunts employ heavily mutagenized P0’s. Backcrossing will also reveal X-lined mutations, as you recall from the previous exercise.

The second parallel task is linkage testing; that is, you will determine which of the five autosomes (linkage groups) your mutation is located on. This is prerequisite to mapping. This 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 "m" represents your recessive mutant tested with reference marker "r"). The markers m and r must be distinguishable. Since homozygous mutant males usually won't mate, the desired trans double heterozygote is constructed by mating males heterozygous for one mutation (m/+) with hermaphrodites homozygous for the reference mutation (r/r). The genotypes of the F1 hybrids will be +m/r+ and ++/r+. We are only interested in the double heterozygote. The F1 hybrids containing only r are not useful. To select the m/r heterozygotes, we clone 4 or 5 individual F1's on small plates. We score the progeny of the F1 individuals (the F2) for linkage. Only F1 clones which segregate m/m homozygotes are scored. This should be 50% of the plates.

F2 progeny of each class are counted: m, r, mr double, and wild-type. If assortment is independent, progeny will be: 9/16 wild type; 3/16 r, 3/16 m; 1/16 rm (that is the 9:3:3:1 ratio)!

On the other hand, if the markers are closely linked, phenotypic ratios will approximate 1:2:1, owing to meiotic segregation of the trans double heterozygote

To Do Today

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

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!