WEEK 3 YEAST RESEARCH PROJECT

Attendance: You will attend two sessions (Tuesday/Thursday OR Wednesday/Friday) in

both weeks 3 and 4.

Project: Each student will be provided with a strain of the yeast Saccharomyces
cerevisiae. http://www.yeastgenome.org/ is a general resource you may wish to explore as
the project proceeds.

They will be expected to:

Produce a liquid culture of these yeast cells, extract total DNA, (week 3) and use
PCR (week 4) to characterize the DNA sequences around the Open Reading
Frames entitled: SBA1 and CPR.

Use colony formation on different types of agar plates to phenotypically analyse

the same yeast strain (week 4).

Use cell counting and agarose gel analysis to estimate how much DNA there is in
a haploid yeast cell (week 4).

PREPARATION FOR WEEK 3:

Theoretical: You will need to be able to:
Understand the basic yeast cell cycle
http://www.csb.ethz.ch/research/dynamic/img/Scer-cellcycle?hires

and lifecycle.

http://tolweb.org/onlinecontributors/app?service=external/ViewImageData&sp=35605

Understand how to use an autoclave to sterilise liquids.

https://www.youtube.com/watch?v=2k75J_uiPD0

Understand how to extract DNA from yeast cells e.g.

http://mckeogh.googlepages.com/YeastGenomicDNAextract.pdf.
http://www.protocol-online.org/biology-forums/posts/19484.html

You will be expected to be able to:

Use a balance, spectrophotometer and micro-pipettes. Revise your lab sessions form first
year and watch this video on micropipettes http://www.youtube.com/watch?
v=uEy_NGDfo_8
Make simple solutions from powder OR stock solutions. If you are unsure about these
calculations you might find the following links useful:

Basic calculations and ratios. Select the topics that interest you here:
https://www.khanacademy.org/math/arithmetic/fractions/understanding_fractions/v/introd
uction-to-fractions
https://www.khanacademy.org/math/arithmetic/rates-and-
ratios/ratios_and_proportions/v/introduction-to-ratios--new-hd-version

Basic introduction to the mole:

http://www.khanacademy.org/science/chemistry/chemical-reactions-stoichiometry/v/the-
mole-and-avogadro-s-number

Molarity: http://www.youtube.com/watch?v=jxSoR8RJDW0
And http://www.youtube.com/watch?feature=fvwp&NR=1&v=OfnuPEvw38E
AND http://www.youtube.com/watch?feature=endscreen&v=SWoo4wgC6oM&NR=1

Make a serial dilution using sterile technique.

http://www.youtube.com/watch?v=MGwo_JvKzXE&feature=related
http://www.protocol-online.org/biology-forums/posts/22485.html

ENSURE THAT YOU ARE CONFIDENT IN THE USE OF THESE

TECHNIQUES/PROCEDURES BEFORE GOING TO THE LABORATORY.
IN THE LABORATORY:

REMEMBER TO USE YOUR PRACTICAL NOTEBOOK TO:

RECORD ALL VOLUMES AND WEIGHTS.

PROVIDE SUFFICIENT INFORMATION FOR ANOTHER STUDENT TO

DO THE SAME EXPERIMENT.
 SESSION 1.
1. Preparation of solutions:

Prepare 20 ml of 1.0 M sorbitol in water and autoclave it.

You will need to:
Use a glass container why?
Work out how much sorbitol to weigh out, and how to make the solution. ie Do you
simply add the powder to 20ml of water OR is there something you need to be careful
about in this respect?
Write your bench position on the cap and bring your solution to the autoclave on the front
bench. Please bring your notes on how to use a potable bench-top autoclave to the
laboratory session. You may find useful points here:
http://oomyceteworld.net/protocols/autoclave%20operation.pdf
http://www.youtube.com/watch?v=6YVqwnWnGdo&feature=fvwrel

Prepare 1.0ml of 12mM MgCl2 in a microtube (available from the plastic petri dish) for
use in the PCR reaction in week 4. You will need to dilute a concentrated stock solution
of MgCl2 in sterile water to do this. Ensure that you understand the principles behind
this. The molarity of the stock solution will be announced on the day. Label the cap with
your bench position.

Preparation of cultures:
a. Preparing rich YEPD medium:
You have been provided with 20 ml of rich medium.
Using sterile technique add some of the stock glucose solution (50% glucose) to it such
that the resulting YEPD contains 2% glucose.
What volume will you need to use?

b. Diluting the culture:

Shake the yeast suspension provided well and then use sterile technique to make a 20
fold dilution of the stock yeast culture in 5ml of sterile water.
What volumes will you need to use?
Using aseptic technique, remove 1ml and measure the optical density at 600nm.
What will you use as a blank?
Now further dilute the 20 fold diluted suspension using aseptic technique (adding
intermediate dilutions if necessary) such that when it is finally added into the fresh
medium it will have a final O/D of 0.001. Check your dilution plan with a facilitator
BEFORE doing it! Label the freshly inoculated culture and incubate it at 250C.
 SESSION 2.
1. Remove 10ml of your yeast culture into a sterile universal container and store it in the
fridge.

2. Extract and precipitate Yeast DNA from the remaining cells as follows:

NB SOLUTIONS CAPABLE OF EXTRACTING DNA

FROM YEAST CELLS CAN ALSO DISSOLVE YOUR
CELLS!
THE ON-LINE EXAMPLE USES PHENOL/CHLOROFORM WHICH ARE VERY
CORROSIVE. WE ARE USING A KIT TO AVOID USING THESE. THE KIT
CONTAINS RELATIVELY HARMLESS CHEMICALS WHEN USED CORRECTLY

PLEASE

1. WEAR GOGGLES, GLOVES AND A FASTENED WHITE COAT.

2. REFRAIN FROM USING PHONES AND OF COURSE EATING/DRINKING
WHILST IN THE LABORATORY.
3. DO NOT DEAL WITH SPILLAGES YOURSELF. REPORT ALL SPILLAGES
TO THE TECHNICAL STAFF.
4. REMOVE GLOVES AND WASH HANDS BEFORE LEAVING THE LAB
EVEN FOR A SHORT BREAK.
5. FOLLOW THE INSTRUCTIONS CAREFULLY.

INSTRUCTIONS

1. Carefully balance a plastic centrifuge tube of your yeast cells in the centrifuge
against that of one of your colleagues.

CHECK THIS WITH A FACILITATOR BEFORE CONTINUING.

2. Turn on the centrifuge at top speed for 1 minute.

3. Decant the liquid medium into the pink liquid in the waste container and then
resuspend the cell pellet in 1ml of sorbitol (you prepared this in the last session)
and transfer the entire suspension to a 1.5 ml micro-centrifuge tube.
4. Pellet cells by centrifugation in a microfuge at 3,000-5,000 g for 30 secs.
Discard the supernatant as in 3.

5. The extraction solutions are in the coloured tubes see the bench for details.
Suspend cells in 800l of the Y-PER Reagent. Mix by gently vortexing or
inverting the tube or pipetting up and down until the mixture is homogenous.

6. Incubate at 65C for 9 minutes.

7. Centrifuge at 13,000 g for 4 minutes, discard supernatant, add 400 l of DNA

Releasing Reagent A, and 400 l of DNA Releasing Reagent B to the pellet. Mix
to produce a homogenous mixture and incubate at 65C for 9 minutes.

8. Add 200 l of Protein Removal Reagent to this mixture and invert several times.
Centrifuge at 13,000 g for 5 minutes and CAREFUL HERE - transfer THE
SUPERNATANT to a new 1.5 ml centrifuge tube. DISCARD THE TUBE AND
PELLET INTO THE BIN PROVIDED.

9. Add 600 l isopropyl alcohol to fill tube containing the SUPERNATANT. Mix
gently by inversion. Precipitate genomic DNA by centrifuging the mixture at
13,000 g for 8 minutes.

10. Remove the supernatant, being careful not to discard the pellet, which is clear and
difficult to see.

11. Add 1.5 ml 70% ethanol to the pellet, invert several times and centrifuge at
13,000 g for 1 minute to wash off any residual salt. ( If there is less than 15
minutes left skip step 12.)

12. Decant this and then repeat step 11 to completely remove any remaining salt.

13. Decant the solution, invert the tube and allow to dry

14. Resuspend in CAREFUL HERE 50 l sterile water. WHICH PIPETTE DO

YOU NEED? Flick the bottom of the tube carefully, or pipette solution up and
down. The pellet should solubilise completely within minutes.

15. Store in the freezer until next week.