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7.01x Lab #3
Mutant Hunt
The main idea: Geneticists use mutants to figure out how a biological system is
supposed to work. First you identify a trait (phenotype) that you are interested in
studying. Then you find an individual that does not have that trait. If you can then figure
out which gene is different in your mutant, you have found the gene that is responsible
for the trait.
A. How are genes responsible for traits?
In this set of experiments we are interested in studying the ability of the yeast
Saccharomyces cerevisiae to make the amino acid cysteine.
B. Why would we be interested in cysteine biosynthesis?
C. Why would we choose to study this process in yeast?
Stage 1: Making Mutants
We did this stage the first week.
A yeast culture was grown up overnight in SC liquid medium at 300C with vigorous
shaking.
A. Prepare cells for irradiation
1. Dilute the yeast culture 1:10 in water in a 1.5ml centrifuge tube using the
appropriate size pipetmen and tips.
10l of overnight culture
90l of H2O
100l of 1:10 dilution
2. Using a pipetman and tip, place 10l of the 1:10 dilution under the cover slip of
the hemacytometer.
3. Using the microscope, count the number of cells in a 4x4 quadrant.
4. Multiply that number by 2.5x105 times the dilution number (in this case is 10).
Number of cells/ml = 43 x 10 x 2.5 x 105 = 1 x 108
How do we decide how many colonies to plate?
5. Calculate the dilution of cells need, so that 200l will contain 750 cells (we are
aiming for irradiation that will yield approximately 33% survival, giving ~250
colonies per plate when we plate 200l).
6. When deciding how much of the final dilution is needed, keep in mind that we
will be plating 200 plates and each requires 200l of cell mixture. Dilute the cells
to the appropriate concentration. This will probably require a series of dilutions.
Make sure that you have a little extra of the final dilution because some liquid
will get stuck to the tube and the tips.
10l of overnight culture 200l of 1:100 dilution 16.9ml of 1:104 dil.
990l of H2O 19.8mls of H2O 28.1l of H O
2
1ml of 1:100 dilution 20ml of 1:104dilution 4
45l of 1:2.7x10 dil
B. Irradiate
7. Pour the diluted culture into a sterile 10cm Petri dish and replace the lid. (use
11mls per dish and multiple dishes if required).
8. Add a sterile stir bar into the dish.
9. Place the dish on the stir plate under the UV source and remove the lid. Start
the stir bar spinning medium speed (not so fast that the liquid will splash).
10. Put on protective eye wear , close the curtain, and turn on the UV source. It
will flicker for a few seconds and then shine steadily. Once the flickering has
stopped, turn on the timer. After 90 seconds, turn the UV source off.
C. Plate
11. Aliquot the irradiated cells into tubes for easier plating and so that they will
remain uncontaminated.
12. Label the Petri dishes with the plate number, your initials, and the date.
Always label plates on the bottom because lids can easily get mixed up.
13. Add a few (3-8) sterile glass beads on top of the agar in each plate.
14. Using a pipetman and tip, transfer 200l of the irradiated cells onto each plate
on top of the beads. Holding the plates level, shake them back and forth in all
directions to spread the liquid around evenly. Shake for approximately 30
seconds.
15. Pour the beads into the bead recycling can.
16. Place the plates in a cardboard box (to keep the light out) and incubate at 300C
for 3 days.
Stage 2: Screening for Mutants
We are going to perform this step today.
Yeast have been mutagenized and grown for 3 days on complete medium agar. At 300C
as described in Stage 1.
A. What is in complete medium?
B. What is in minimal medium?
C. If you wanted to make media on which to grow a strain that was unable to synthesize
cysteine, what would you need to put in it?
D. What medium could we use to screen for cysteine auxotrophs?
E. What is a selective condition?
F. Why don’t we plate the mutagenized yeast directly on selective conditions?
G. How should we find the rare mutant yeast that cannot synthesize cysteine within the
large collection of yeast colonies we have on the plates?
Replica plating protocol:
1. Obtain 3 plates that have mutagenized yeast grown on them. Label them 1-3 and with
your initials.
2. Obtain 3 CM plates (no stripe), 3 CD plates (blue stripe) and 3 MM plates (red stripe).
3. Label the bottom of each set of the fresh plates with your initials, the appropriate 2
letter code and number them 1-3.
4. Take the replica plating block and remove the ring. Carefully place a velvet on top of
the block, smooth side up. Be careful to only touch the edges of the velvet or else you
will contaminate your plates.
5. Place the ring back down over the block so that it pulls the velvet tight and smooth
across the top of the block.
6. Take plate number 1 that has yeast growing on it. Remove the lid and invert the plate.
Gently place it down on the velvet and tap the entire surface with your fingers so that the
yeast colonies will transfer some cells to the velvet. Do not press very hard as that will
smush the colonies.
7. Lift the plate off of the velvet and replace the cover.
8. Remove the cover from CM plate #1, invert the plate and gently place it on the velvet.
Repeat the tapping procedure to transfer some cells onto the fresh plate. Mark the
bottom of the plate with a little line to indicate where the mark on the tape was on the
ring that holds the velvet in place. This will be used to determine the orientation of the
plate.
9. Remove the plate by lifting it directly up and replace the lid.
10. Repeat the transfer process (Steps 8 and 9) with CD plate #1 and MM plate #1.
11. Remove the ring and the velvet from the replica block and repeat steps 4-10 with
plates #2 and 3.
12. Invert all plates (bottom side up) and place at 300C overnight.
Stage 3: Identifying Mutants
We are going to perform this stage today with plates that are replica plates (replica
plating done by Dina) of the yeast that were plated the first week of class.
A. When you “screen” the plates, how do you identify the mutants?
B. Mutant Screening Protocol:
1. Get the stack of plates that is labeled with your initials.
2. Open the plastic sleeve and carefully invert the stack of plates.
3. Gently pull off the plastic sleeve being very careful not to knock over the stack
of plates.
4. There are 3 kinds of plates in the stack. Unmarked plates contain complete
media (CM), plates with a blue stripe contain cysteine dropout media (CD), and
plates marked with a red stripe contain minimal media (MM). Please label the
bottom of each plate with the appropriate two letter code.
5. Look for cysteine auxotrophs: Compare the colonies on the CM plate with the
colonies on the CD plate. Are there any missing from the CD plate?
There are a few methods for comparing the plates:
a) Pattern recognition: Examine the shape of a group of colonies on the CM plate
and then look for that group of colonies on the CD plate. Make sure that none of
the colonies are missing.
b) Place the CD plate on top of the CM plate with the bottom sides of the plates
facing you. Look through the CD plate and see if there are any colonies on the
CM plate that do not have a corresponding (covering) colony on the CD plate.
c) Look for “halos”: areas where the cells have been transferred but have not
grown. These look like very faint colonies.
If you find a colony on the CM plate that did not grow on the CD plate, draw
a circle around that colony on the bottom of the CM plate and show one of
the instructors.
6. Look for amino acid auxotrophs. Repeat the process in step 5 but using the
CM and MM plates.
C. If we find some colonies that grow on CM but not MM, can we determine which
amino acid they are unable to synthesize?
D. How might we figure this out?
Thought question:
1. In 1908 a physician in London named Archibald Garrod had some new patients with
an unusual condition: when their urine came into contact with air it turned black.
Compare his quandary to the lab we did today.
In both cases (our lab and Dr Garrod’s clinic) who or what
Is the phenotype?
Is the assay?
Is the mutant?
By calling something or someone a mutant what assumption have we made?
What else would you want to know about Dr Garrod’s patients to determine if this
assumption is correct?
