Protocols

A selection of general and specialised protocols provided by Kent Fungal Group

S.cerevisiae tranformation

Saccharomyces cerevisiae LiAC transformation

Protocol is for one transformation. Multiply by number of transformations to be done.

1. Grow cells to mid to late log phase (OD600 0.5-0.6). 1 ml per transformation is plenty.

2. Harvest cells in clinical centrifuge. Wash once with 1 ml TE and once with 1 ml LiOAc in TE.

3. Resuspend in 0.1 ml of LiOAc in TE.

4. Add 15µl of carrier DNA. (1 mg/ml Single stranded DNA boiled 15’ and iced)

5. Add 0.1 - 1µg transforming DNA and mix gently.

6. Add 700 µl 40% PEG4000 in 0.1M LiOAc in TE. Vortex.

7. Incubate with rotation for at least 1 hr. at room temperature on roller.

8. Incubate for 15 min at 42°C.

9. Spin down and resuspend in 200ul sterile H2O. Plate on selective plates.

Inoculate one colony in 100 ml YPD and grow to OD 1.3.
Harvest at 5000 rpm for 2 minutes
Wash in 25 ml sterile H2O
Resuspend in 25 ml TELiDTT (using fresh DTT). Incubate at room temperature for 1 hour.
Centrifuge at 5000 rpm and 4 °C for 5 minutes, wash in 25 ml ice-cold water, 10 ml ice-cold 1M sorbitol.
Transfer to a cold 15 ml tube and centrifuge at 5000 rpm and 4 °C for 5 minutes. Resuspend in 100 µL ice-cold 1M sorbitol. Keep on ice.
Electroporation:

For each transformation put 40 µL of cells in a 1.5 ml Eppendorf tube.

Add 500 ng of DNA

Incubate on ice for 5 minutes.

Transfer mixture to an electroporation cuvette

Place in electroporation chamber and pulse at 1.5 kV, 25 µFD and 200 ohms.

Remove cuvette and immediately add 1 ml 1M sorbitol, mix by inversion.

Plate 100 µL on SD-Ura with 1M sorbitol.

Solutions:

TELiDTT (100ml)

Filter sterilise

FACS Analysis of Propidium Iodide Stained Cells to Assess DNA content

Spin down 1 x10^7 cells (about 100µl of an overnight culture) and remove supernatant.
Add 1 ml of ice cold 70% Ethanol and vortex (cells will appear clumped, this is normal), leave for 10 mins (this stage fixes the cells by dehydration).
At this stage these cells can be stored at 4C indefinitely if required (good for time courses)
Spin down cells, remove ethanol solution and re-hydrate by adding 1 ml of 50mM Na Citrate.
Spin down again, discard supernatant and resuspend pellet in 0.5ml of 50mM Na Citrate containing 1mg/ml RNAase A (as PI binds to all nucleic acids, this step removes the background by digesting RNA in the cell) and incubate for 2h at 37oC.
Add PI to a final concentration of 6µg/ml.
Load entire contents of the tube into a FACS tube and record fluorescence using the FL2 (red) detector.

Western blotting

Reagents

Transfer Buffer: into a 1 liter bottle, weigh 2.2 g of Tris, 13.5 g of glycine and 0.9 g of SDS. Add 500 ml of water and stir till the solids are dissolved. Add 135 ml of methanol, then fill up to 900 ml with water.
10x TBS: 500 mM Tris pH 7.8, 1.5 M NaCl.
TBS-M: For 100 ml, mix 10 ml of 10x TBS, 5 g of Marvel or another dried milk powder, and add water to 100 ml. Stir until the Marvel is dissolved. You will need about 100 ml per blot.
TBS-T: For 100 ml, mix 10 ml of 10x TBS, 50 μl of Tween 20, and add water to 100 ml. Stir until the Tween is dissolved. You will need about 100 ml per blot.
PVDF Membrane.
Blotting paper.
Primary Antibody specific to the protein to be detected.
FITC-labelled Secondary Antibody compatible with the primary antibody, eg anti-rabbit (eg Sigma F9887), anti-mouse (eg Sigma F2012) or anti-goat (eg Sigma F7367).

Cut a piece of PVDF membrane to exactly fit the size of the gel (8.2x5.0 cm for BioRad Protean III mini gels).
When the SDS-PAGE gel that is to be transferred has run, remove it from the running assembly and incubate it together with the PVDF menbrane in transfer buffer for 10 minutes or longer.
Cut two pieces of blotting paper to the same size as the membrane.
Assemble the blot: soak one piece of paper in transfer buffer, place it flat on the lower electrode of the semi-dry blotter. Place the soaked PVDF membrane on this (it should exactly overlay the piece of paper), the gel on this, and the second piece of paper (also soaked in transfer buffer) on top. Roll an empty plastic tube or rubber roller over the sandwich to expell any air bubbles. Place the upper electrode on top of the apparatus.
Blot at 9V, 200 mA for 30 minutes.
When the run has finished, remove the PVDF membrane from the sandwich and incubate in TBS-M for 10 minutes or more.
Dilute the primary antibody into 15 ml of TBS-M. Incubate the membrane in this over night at 4 C.
Dilute the FITC-labelled secondary antibody into 15 ml of TBS-M. Rinse the mebrane once with TBS-M, then incubate with the scondary antibody for 1 hour at room temperature.
Wash the membrane four times with TBS-T (~1-2 minutes per wash).
Place the membrane on a piece of dry, clean paper, cover with a small box to protect from light and leave for 20-30 minutes to dry.
Scan the membrane in the Fuji FLA5100 scanner using the FITC settings.

C.albicans transformation

FACS

Western blotting