DATE 03/30/2010 EXPERIMENT - 10

YEAST MATING
PURPOSE OF THE EXPERIMENT

The aim of the experiment is to carry out yeast mating to create

diploid cells by introducing pGBKT7-53 of AH109 and pGADT7-T

plasmids into the same host cells. These colonies created by diploid

cells were then screened by PCR in the next experiment.

REAGENTS/MATERIALS

• YPD medium

• SD Agar plates containing -Leu/-Trp which is double dropout

• SD Agar plates, -L/-W/-H/-A +X-α-gal (QDO)

• AH109 (pGBKT7-53) bait vector transformant

• Y187 (pGADT7-T) which is a prey vector transformant

• 1.5 ml tubes

• LB broth

• 37°C Incubator

PROCEDURE

DAY- 1:

REHYDRATION OF DRY STRIP :

• This part of the experiment was carried out by the TA.

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 • 125 μl of re-hydration buffer which is composed of 8 M urea, 2%

CHAPS, 2% IPG Buffer, 0.002% bromophenol blue was pipette

into the IPG Reswelling Tray in a flat level bench.

• Care was taken to check that the volume does not exceed the

maximum volume determined for each Immobiline DryStrip size

which is 125 μl for 7 cm IPG strip.

• The foil cover is carefully removed from the DryStrip and the

strip is carefully placed in the tray channel with the gel side

down.

• The strip was then overlayed with 400 μl of mineral oil to prevent

evaporation and rehydrated overnight at the room temperature.

DAY- 2

FIRST DIMENSION / IEF

• The samples were in solution and then first dimension run was

prepared.

• All the connections were properly checked and was set up

carefully.

• The temperature on MultiTemp III Thermostatic Circulator

cooling unit was set to 20 °C before 30 min and the cooling plate

was placed in Multiphor II Electrophoresis unit.

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• 4ml of paraffin oil was pipette onto the cooling plate and the

Immobiline DryStrip Tray and Electrode Holder were carefully

positioned on the cooling plate.

• The red electrode is connected connection and care was taken

that there are no larger bubbles between the tray and the plate.

• Both anode and cathode were connected to Multiphor II Unit.

• 10 ml of paraffin was poured into the tray and Immobiline

DryStrip Aligner having 12 grooves side up was placed into the

tray on top of the oil.

• Two IEF Electrode Strips which were prepared from 2 mm thick

filter paper were cut to a length of 11 cm each and placed onto a

clean flat surface and each one was soaked with 0.5ml dH2O.

• The rehydrated IPG strips were then transferred and the

electrodes were put on the IEF electrode strips.

• The rehydrated IPG gel strips were rinsed with deionized water

and were placed on a sheet of water saturated filter paper.

• A second sheet of filter paper was made wet with deionized

water and placed onto the surface of the IPG gel strips. These

were blotted gently for a few seconds to remove excess

rehydration solution.

• The rehydrated IPG strips were then transferred to adjacent

grooves of the aligner in the tray.

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• The strips with the acidic end were placed at the top of the tray

near the cooling tubes and anode while the cathodic end should be

at the bottom of the tray near the cathode.

• The strips were aligned such that the anodic gel edges are lined

up and the moistened IEF Electrode Strips were then placed on

top of the aligned strips at the Cathode and Anode.

• Electrodes were pressed down on top of IEF Electrode Strips.

• One sample cup was placed above each rehydrated IPG strip and

finally press down the sample cups to ensure good contact with

each strip.

• 80 ml mineral oil of poured after the sample cups were properly

positioned to ensure that IPG gel strips were completely covered.

• Each sample cup was filled with a few drops of DryStrip Cover

Fluid to completely cover the sample cup.

• Protein samples (up to 100 µl – 20 µl; max. 10 mg/ml) were placed

into the cups by pipetting under the surface of the DryStrip

Cover Fluid to make sure that the sample was totally sunk to the

bottom of the cup.

• Lid of the Multiphor II electrophoresis chamber was closed and

checked if it was correctly assembled.

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 • The leads were connected on the lid to the power supply and
power was set up as below:

Phase V mA W Duration kVh

(h:min)
1 200 2 5 0:01 0.001
2 3500 2 5 1:30 2.8
3 3500 2 5 0:35-1:05 2.2-3.7
Total 2:05-2:35 5-6.5

• The entire set up was dismantled and the IPG gel strips were
stored between two sheets of plastic film at -80°C.

Second Dimension/SDS-PAGE:

Part – A

Chemicals and Buffers:

• SDS

• Iodoacetamide

• DTT

• Tris

• Bromophenol blue

• Glycerol

• Urea.

• Resolving gel buffer which is composed of 1.5 M Tris-HCl, pH

8.8 and 0.4% (w/v) SDS

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 • 500ml of Equilibration buffer that was prepared by adding 180

g of urea, 150 g of glycerol, 10 g of SDS and 16.7 ml of

resolving gel buffer. This was dissolved in dH2O and filled up

to 500 ml.

• 10ml Bromophenol blue solution that was made by dissolving 25

mg of Bromophenol blue in 10 ml of resolving gel buffer. This

was stored at 4°C.

• SDS Running buffer which is composed of 25 mM Tris, pH 8.3,

192 mM glycine, 0.1% SDS.

• 0.5% agarose in 1X running buffer.

B. Equilibration of IPG strips

• 100 mg of DTT was dissolved in 10 ml of equilibration buffer - I

• IPG gel strips that were taken out from the freezer were placed

into individual test tubes.

• 10 ml of equilibration buffer I and 50 µl of the Bromophenol Blue

solution were added and the test tubes were sealed with

Parafilm.

• These were then shaked for 15 min at RT and equilibration

buffer I was then poured off.

• 400 mg of iodoacetamide was dissolved in 10 ml of equilibration

buffer- II .

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 • Equilibration buffer II and 50 µl of Bromophenol Blue solution

were then added to the test tubes and equilibrated for another

15 min on a shaker at RT.

• IPG gel strips were then rinsed with dH2O for a second and

placed on a piece of filter paper at one edge for a few minutes

for the excess equilibration buffer to get drained off.

C. SDS-PAGE

• Prepare a 8% resolving gel was prepared with two-well comb

having 0.75 mm thickness.

• dH2O was overlayed onto the surface of the gel after pouring.

Percent ddH2O 30% Gel1.5M Tris- 10% SDS

Gel acrylamide HCl buffer

(pH 8.8)

8% 4.7 mL 2.7 mL 2.5 mL 0.1 mL

• BioRad electrophoresis apparatus was set up.

• The equilibrated IPG gel strips were immersed in SDS running

buffer for a few seconds and excess buffer was removed.

• Rinse the SDS Gel with SDS Running Buffer and place the IPG

gel strip having 0.5 mm thickness on the top surface of an SDS

gel

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• This IPG strip was pressed with a thin spatula onto the surface

of the SDS gel to ensure that it is in complete contact.

• The top surface was overlayed with hot agarose which is 0.5% in

1X running buffer.

• The agarose was allowed to solidify for 5 min.

• 5 μl of marker was loaded in another small lane.

• The gel was run at 200 V for about 35 minutes or until the dye

reaches the bottom of the gel. Then, gel was carefully pealed off

the glass plate and placed in a tray containing Gel Code Blue

solution.

• Picture of the gel was taken and analyzed.

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