GENERAL PROTOCOLS AND REAGENTS

II. GENERAL PROTOCOLS AND REAGENTS

11.1. Plasmid DNA Isolation

Small-scale Preparation ofPlasmid DNA

1. Inoculate 2 ml of LB-amp media (rich media supplemented with 75 f.lg/ml

ampicillin or 25 f.lg/ml kanamycin or 170 f.lg/ml chloremphenicol) with a small

amount of cells (1 mm colony size). Incubate in an orbital shaker at 37°C for 16 hrs

with shaking at 220 rpm.

2. Vortex the tubes briefly and transfer cultures to sterile 1.5 ml microfuge tubes Spin

down at 6000 rpm for 5 min at RT. Decant supernatant and discard. (Optional:

Wash cell pellet with 500 f.ll STE buffer (114th volume of culture).

3. Add 100 f.ll of ice-cold solution I. Vortex vigorously to resuspend cells.

4. Add 200 f.ll of solution II. Mix by inverting the tube rapidly five times (Turbidity

disappears because of cell lysis). Incubate on ice for 5 min. (Do not vortex.)

5. Add 150 f.ll of ice-cold solution III. Mix thoroughly by slow inversion. Incubate on

ice for 5 minutes.

6. Spin down bacterial lysate at 13,000 rpm for 10 min at 4°C in a refrigerated

microcentrifuge. Collect supernatant in a fresh microfuge tube containing 900 f.ll of

chilled 100% ethanol. Mix thoroughly by inversion. Incubate tubes at -20°C for 4

hours or -80°C for 1 hr.

7. Centrifuge at 13,000 rpm for 15 min at 4°C in a refrigerated microcentrifuge to

pellet down plasmid DNA. Decant supernatant.

8. Add 1 ml of chilled 70% ethanol. Rinse walls of tube by inverting it several times.

Spin again under the same conditions for 10 min. Decant supernatant. Give a brief

24

spin and aspirate traces of ethanol with a pipette. Leave the tube with open lid at

37°C for 10 min to dry out DNA pellet.

9. Resuspend in 30 J.ll of0.1x TE/4 J.lg RNaseA (10 mg/ml). Mix by tapping. Incubate

at 37°C for 1 hr. Check the quality of DNA preparation and presence of RNA in the

sample by agarose gel electrophoresis. This DNA preparation can be used in test

reactions for restriction digestion for the purpose of screening bacterial

transformants. Purified DNA is preferred for large scale digestions. Proceed to step

10 onwards for purification ofDNA.

10. Increase volume of DNA preparation from 30 J.ll to 100 J.ll with water. Add equal

volume ofPCI mix (phenol: chloroform: isoamyl alcohol as 25:24:1). Vortex for 1

min and centrifuge at 10,000 rpm for 8 minutes at room temperature (RT). Two

layers can be observed. Collect upper aqueous layer into a fresh tube.

11. Re-extract the organic layer with 50 J.ll of 1x TE. Vortex for 1 min and centrifuge at

10,000 rpm for 8 minutes at RT. Collect and combine the upper layer with the

preVIous one.

12. To this aqueous layer, add equal volume of chloroform-isoamyl alcohol mix (24:1).

Vortex for 30 sec and centrifuge with same settings as in step 15. Collect upper

layer in fresh tube.

13. Add 1/10th volume of 3 M sodium acetate, 0.002% linear acrylamide (0.25% w/v)

and 2.5 volumes of chilled 100% ethanol. Mix thoroughly by inversion. Incubate at

-20°C for 4 hours or -80°C for 1 hr.

14. Repeat steps 8 and 9 (Addition ofRNase can be omitted if RNA is not seen on gel

at step 9.).

15. Analyze DNA preparation (and presence of RNA, if any) on agarose gel

electrophoresis. Also, quantitation of DNA (after it has been linearized) can be done

25

by comparing it with a known amount of DNA on agarose gel. Label tube

mentioning plasmid DNA name, concentration and date of preparation. Store at -

Large-scale Preparation of Plasmid DNA

1. Inoculate 25 ml of LB-amp media (rich media supplemented with 75 J.lg/ml

ampicillin or 25 J.lg/ml kanamycin or 170 J.lg/ml chloremphenicol) with a small

amount of cells (1 mm colony size). Use 250 ml conical flasks for growing the

cultures. Incubate in an orbital shaker at 37°C for 16 hrs with shaking at 220 rpm.

2. Transfer cultures to two sterile Oakridge tubes (Tarsons or Polylab ). Spin down at

4000 rpm for 10 min at 4 °C. Decant supernatant and discard.

3. Wash cell pellet in each tube with 3.125 ml STE buffer (1/4th volume of culture).

Spin down at 4000 rpm for 10 min at 4°C. Decant supernatant and discard.

4. Add 400 J.ll of ice-cold solution I to each tube. Vortex vigorously to resuspend cells.

Split them into two microfuge tubes so that there are a total of four tubes at this

step.

5. Add 400 J.ll of solution II to each tube. Mix by inverting the tube rapidly five times

(Turbidity disappears because of cell lysis.) Incubate on ice for 5 min. (Do not

vortex).

6. Add 300 J.ll of ice-cold solution III. Mix thoroughly by slow inversion. Incubate on

ice for 5 minutes.

7. Spin down bacterial lysate at 13,000 rpm for 10 min at 4°C in a refrigerated

microcentrifuge. Collect supernatant in a fresh microfuge tube.

8. Add 500 J.ll isopropanol to precipitate out DNA. Mix thoroughly by inversion.

Incubate tubes at RT for 30 min to 1 hr.

9. Centrifuge at 13,000 rpm for 15 min at RT in a refrigerated microcentrifuge to

pellet down plasmid DNA. Decant supernatant.

26

10. Add 1 ml of chilled 70% ethanol. Rinse walls of tube by inverting it several times.

Spin again with the same conditions for 10 min to recover DNA. Decant

supernatant. Give a brief spin and aspirate traces of ethanol using pipette.

11. Leave the tube with open lid at 37°C for 10 min to dry out DNA pellet.

12. Resuspend in 100 J.!l of 0.1x TE/4 Jlg RNaseA (10 mg/ml). Mix by tapping.

Incubate at 37°C for 1 hr. Pool the contents of the four tubes into one. Check the

quality of DNA preparation and presence of RNA in the sample by agarose gel

electrophoresis.

13. Add equal volume of PCI mix (phenol: chloroform: isoamylalcohol as 25:24:1).

Vortex for 1 min and centrifuge at 10,000 rpm for 8 minutes at RT. Two layers can

be observed. Collect upper aqueous layer in fresh tube.

14. Re-extract the organic layer with 100 J.!l of 1x TE. Vortex for 1 min and centrifuge

at 10,000 rpm for 8 minutes at RT. Collect and combine upper layer with the

previous one.

15. To this aqueous layer, add equal volume of chloroform-isoamylalochol mix (24: 1 ).

Vortex for 30 sec and centrifuge with same settings as in step 15. Collect upper

layer in fresh tube.

16. Add 1/lOth volume of3 M sodium acetate, 0.002% linear acrylamide (0.25%) and 2

volumes of chilled 100% ethanol. Mix thoroughly by inversion. Incubate at -20°C

for 4 hours or -80°C for 1 hr. Repeat steps 10 to 12. Resuspend DNA in 50 J.!l O.lx

TE.

17. Analyze DNA preparation (and presence of RNA, if any) on agarose gel

electrophoresis. Also, quantitation ofDNA (after it has been linearized) can be done

by comparing it with a known amount of DNA on agarose gel. Label tube

27

mentioning plasmid DNA name, concentration and date of preparation. Store at -

Reagents for Plasmid DNA Preparation

Note: Use double-distilled or deionized water for making reagents.

1) STE buffer, 20 ml

Composition Volume

Tris-HCl, pH 8.0 200 J.!l EDTA 40 J.1l Sodium chloride (NaCl) 400 J.1l Water make up to 20 ml

Final

lOmM lmM

lOOmM

Use autoclaved components to make solution. Store at 4°C.

2) Alkaline Lysis Solution I, 40 ml

Composition Volume Final

Tris-HCl, pH 8.0 l.Oml 25mM EDTA 0.8ml lOmM Glucose 2.0ml 50mM Water make up to 40 ml

Use autoclaved components to make solution. Store at 4°C.

3) Alkaline Lysis Solution II, 10 ml

Composition Volume

Sodium hydroxide (NaOH) 0.2 ml Sodium dodecyl sulphate (SDS) 1.0 ml Water make up to 10 ml

Final

0.2N 1%

This solution is freshly prepared just before use. Use at RT.

4) Alkaline Lysis Solution III, 50 ml

Composition Volume Final

Potassium acetate (CH3COOK) 30.00 ml 3.0M Glacial acetic acid (CH3COOH) 5.75 ml Water make up to 50 ml

Use autoclaved components to make solution. Store at 4°C.

28

Stock

l.OM 0.5M 5.0M

Stock

l.OM 0.5M l.OM

Stock

ION 10%

Stock

5.0M

11.2. Bacterial transformation

Preparation of Electrocompetent cells

Work in sterile (aseptic) conditions as much as possible. Use chilled reagents and plasticware or glassware wherever required.

1. Inoculate 10 ml of LB medium with isolated single colony from freshly grown

bacterial cells (DH10b) on LB plate. Incubate in an orbital shaker at 37°C for 16 hrs

with shaking at 220 rpm.

2. Determine the cell density of pre-culture by measuring the OD600 of 1:50 diluted

culture. Dilute the pre-culture into 500 ml of secondary culture medium (LB

medium) such that the OD6oo of starting culture is 0.06. Incubate in an orbital

shaker at 37°C with shaking at 220 rpm.

3. When OD600 of the secondary culture reaches 0.5, chill the cells on ice for 20 min

with intermittent swirling. Transfer the cultures to sterile and chilled centrifuge

bottles kept on ice-water bath. Spin down at 8000 rpm in SS-34 rotor (Sorvall) for

15 min at 4°C.

4. Discard the supernatant and resuspend the cell pellet in sterile and chilled solution

of 10% glycerol (Qualigens) with a volume equal to that of the starting culture, that

is, 500 ml. Resuspend the cells by swirling and maintain temperature close to 4°C.

Spin down for 15 min under similar conditions.

5. Discard the supernatant and resuspend the cell pellet in sterile and chilled solution

of 10% glycerol (Qualigens) with half the volume of the starting culture, that is, 250

ml. Resuspend by swirling and maintain temperature close to 4°C. Spin down for

15 min under similar conditions.

6. Discard the supernatant and resuspend the cell pellet in 20 ml of sterile and chilled

solution of 10% glycerol (Qualigens) (1125th volume of starting culture volume).

Resuspend by swirling and maintain temperature close to 4°C.

29

7. Transfer the cell suspension to sterile and chilled oakridge tubes and spin down

again. Discard the supernatant and resuspend in a total of ~800 J..Ll of sterile and

chilled 10% glycerol (USB).

8. Determine OD6oo of 500-fold dilution of cell suspension. The desired cell

concentration is ~2-3 x1010 cells/mi. [1 OD6oo=2.5 x 1010 cells/ml]. Store the cells in

small aliquots at -80°C.

Protocol for Bacterial Transformation by Electroporation

Work in sterile (aseptic) conditions as much as possible. Use chilled cuvettes. Ensure availability of SOC

components and LB-Amp plates before the experiment

1. Prepare dilutions of a control DNA (closed circular plasmid of known

concentration), Litmus38 and use final DNA concentration of 20 pg for

electroporation. This is included to calculate the transformation efficiency of

electrocompetent cells. Also, prepare appropriate dilutions of the ligation mix

(generally 5x or 1 Ox).

2. Mix the components of SOC medium just before electroporation and maintain them

sterile. Also, keep the electroporation cuvettes ready and chilled.

3. Take out an aliquot of electrocompetent cells and thaw on ice. Resuspend the cells

by gentle tapping with hand. For 1 mm cuvette, use 20 J..Ll cells per reaction, mix

with 1 J..Ll of DNA and incubate the mix on ice for 1 min before pipetting into the

cuvette towards the bottom. Tap the cuvette gently to remove trapped air bubbles.

4. Set the electroporation apparatus (Biorad # Gene Pulser Xcell™ electroporation

system) to the following conditions: 1800 V, 25 J..LF, 200'0.

5. Wipe offthe wet surface of the cuvette and place it in the shock pad. Give one pulse

and observe the time constant. Add 1 ml of SOC medium to the cuvette within one

minute of giving the pulse. This allows for recovery and stabilization of cell

membranes after the pulse.

30

6. Resuspend the cells gently using 1 ml pipette and transfer the suspension to a

sterile, labeled 15 ml tube.Repeat the process for all samples with fresh cuvette for

each.

7. Incubate the cells at 37°C for one hour with gentle rotation (200 rpm).

8. Plate out an appropriate volume (200 J.ll-lml) on LB-ampicillin media and incubate

the plates at 37°C. For control DNA, use 5 J.ll out of 1 ml for spreading on the

plates.

9. Calculation of the transformation efficiency:

Transformation efficiency = Cfu/pg DNA used x 106 x Dilution factor

Reagents for Electroporation

SOB medium, 100 ml Composition

Tryptone 2.00 g Yeast extract 0.50 g Sodium chloride (NaCl) 0.05 mg Potassium chloride (KCl) 1 ml (Stock 250 mM) Water make up to 100 ml

Dissolve the components in -80 ml water. Allow constant stirring. When completely

dissolved, adjust the pH of the medium to 7.0 with 10 N NaOH. Make up the volume

and sterilize by autoclaving. Store the medium at RT.

SOC medium, 5 ml

Composition Volume Stock

SOB medium 4.7ml MgCh 0.1 ml 1M MgS04 0.1 ml 1M Glucose 0.1 ml 1M

Mix the components just before use. Work under sterile conditions.

31

11.3. Transformation of C albicans

The transformation in C. a/bicans strains was done using electroporation method (163).

Briefly, electrocompetent cells were prepared as per protocol and 40 f.ll of the final cell

suspension was mixed with five f.ll (-0.5-1.0 Jlg) of linear DNA fragment to be

transformed. The mixture was incubated on ice for 1 min and electroporated in a 2mm

cuvette using the electroporation apparatus (Gene Pulser Xcell™; Biorad) as per the

following settings: 2500 V, 25 JlF, 200 n. 1 ml of chilled 1M sorbitol was added to the

cuvette immediately. The cells were suspended gently using a 1 ml pipette and

transferred to a sterile, labeled 50 ml tube. 1 ml of YPD liquid media was added to the

cell suspension and incubated at 30°C for 4h with shaking at 200 rpm. Appropriate

volume (about halt) of transformation mix was then plated out onto selective plate and

incubated at 30°C.

Reagents for C. a/bicans Electroporation

Note: Use double-distilled or deionized water for making reagents.

YPDmedium

Lithium Acetate, 1M, pH=7.5, 20 ml

Dissolve 2.04g of lithium acetate (Sigma) in -15ml water on the magnetic stirrer.

Adjust pH to 7.5 using 10-fold diluted glacial acetic acid. Make up volume to 20ml and

sterilize the solution by passing it through 0.22f.lm (Millipore) and store at 4 degree.

lOX TE, pH=7.5, 20 ml

Components

Water 1M Tris-Cl (pH=8.0) 0.5M EDTA (pH=8.0)

Volume

12 ml' 2ml

0.4ml

Final cone.

100mM 10mM

32

Adjust pH to 7.5 using 10-fold glacial acetic acid and sterilize solution by passing

through 0.22J..tm (Millipore) and store at 4 degree.

2M Sorbitol, 40m1

Dissolve 7.288g of sorbitol (Sigma) in -25ml water with mild heating on the magnetic

stirrer. Make up volume to 40ml and sterilize the solution by passing it through 0.22J..tm

(Millipore) and store at 4 degree.

JMDTT, Sml

Dissolve 771mg of DTT (USB) in Sml of double distilled water. Filter sterilize the

solution by passing it through 0.22J..tm filter (Millipore). Aliquot and store at -20°C.

11.4. Protocol for Genomic DNA isolation

The genomic DNA was isolated from -2x108 cells as follows. Briefly, inoculate the

C. albicans strains into 2ml YPD liquid media and incubate at 30°C in a incubator

shaker till the OD6oo reaches 10. Harvest lml culture was in an eppendorf tube at RT,

wash the cell pellet with lml of lxTE buffer (pH=8.0) and resuspend in 0.2ml of smash

buffer. Add 0.2ml of equilibrated phenol (with O.lM TrisCl buffer, pH=8.0) Add acid­

washed sterile glass beads upto the interface between the organic and aqueous layer and

vortex vigorously for 2 minutes. Add 0.2ml each of lxTE buffer (pH=8.0) and

chloroform::isoamyl alcohol and vortex vigorously for 1 minute. Spin at maximum

speed for 5 minutes at RT. Collect the upper aqueous layer in a fresh eppendorff tube.

Add 125J..tl of 10M ammonium acetate and 0.6ml of isopropanol. Spin at maximum

speed for 10 minutes at RT. Wash the pellet with 70% ethanol. Air dry the DNA pellet

and resuspend in 50 J..tl TE+RNaseA (lOOJ..tglml). Incubate at 37°C for 1 h. Check the

integrity and amount of genomic DNA (and presence of RNA if any) on 0. 7% agarose

gel.

33

Reagents for Preparation of Genomic DNA

Note: Use double-distilled or deionized water for making reagents.

Smash and Grab method

Smash buffer, 10 ml

Composition Volume Final Stock

Tris-HCl (pH 8.0) 0.10 ml lOmM l.OM EDTA 0.02 ml 1mM O.SM Sodium chloride (NaCl) 0.20ml 100mM S.OM Sodium dodecyl sulphate (SDS) 1.00 ml 1% 10.0% Triton X-100 2.00ml 2% 10.0% Water make up to 10 ml

Use autoclaved water and plasticware for making solution.

11.5. Screening of Calbicans transformants by PCR Assay

Genomic DNA isolation is followed by amplification of the specific genomic loci to

verify the targeted integration. A standard recipe for PCR followed in all the

experiments is as follows:

The reaction mixture includes 1x Taq buffer with 20 mM (NH4)2S04, 1.5 mM MgCh,

0.2 mM each dNTP, 0.4 J.lM each primer, 0.025 U Taq DNA polymerase, and 2 ng

genomic DNA in a total volume of 10 J.ll. The PCR conditions were as follows: 94°C,

4' (Step1), 94°C, 1' (Step2}, Ta °C, 1' (Step3), 72°C, 1kb/min (Step4), 72°C, 1 0'

(StepS). Steps 2-4 were repeated for 30 cycles.

All dilutions are made in PCR-grade water, which is freshly autoclaved MilliQ purified

water. Fresh dilutions of the template DNA are used for each set ofPCR.

Gel Analysis of PCR products:

PCR products are analyzed on O.Sx T AE-agarose gels. Gel images were captured and

amplicon size was estimated using the Alphaimager Gel doc system.

34

11.6. Protocol for Cell Lysate Preparation

1. Inoculate 5-10 ml of preculture media (rich media or any specific media as per

requirement) with fresh cells from media plate. Incubate in an orbital shaker at

30°C for 16-18 hrs with shaking at 220 rpm.

2. Determine the cell density of pre-culture by measuring the OD6oo of 1:50 diluted

culture. Dilute the preculture into 50 ml secondary culture media with starting

OD6oo of0.15. Incubate in an orbital shaker at 30°C with shaking at 220 rpm.

3. Harvest the cells after 5 hrs by centrifugation at 5000 rpm for 6 min at 4°C.

4. Wash the pellet with 2 ml of ice-cold water. Suspend the cells in -250 J..Ll of

extraction buffer (215) containing protease inhibitors and transfer the cell

suspension to cold microcentrifuge tubes.

5. Add chilled acid-washed autoclaved glass-beads upto 3 mm from liquid surface.

Vortex for 8 cycles of 1 min vortexing followed by 1 min on ice. Examine cells

under a microscope to determine the extent of cell lysis. It should be -80-90%.

6. Aspirate out the lysate using a wide-bore micropipette tip (ends to be cut-off

straight with a scalpel blade). Spin down for 10 min at 13000 rpm at 4°C to pellet

down the debris. Aspirate out the cleared lysate into a fresh tube and store at -80°C

in small aliquots to avoid repeated freeze-thaw.

The protein concentration was estimated using Bio-Rad protein assay dye reagent.

II. 7. Western Blotting Protocol

Gel analysis of the Extract

For most of the experiments, 8-10% gels of 1.5 mm thickness were used. The

recipe for resolving gel was as follows. 30% (w/v) stock Acrylamide-bis

acrylamide mix (29:1), fmal concentration of 10% or 8%, 0.39 M Tris-HCl, pH 8.8,

0.1% (w/v) Ammonium persulfate, 0.04% (v/v) TEMED. The recipe for stacking

35

gel was as follows. 30% (w/v) stock Acrylamide-bis acrylamide mix (29:1), final

concentration of 5%, 0.127M Tris-HCl, pH 6.8, 0.1% (w/v) Ammonium persulfate,

0.1% (v/v) TEMED.The cell extracts (about 20-100 f..lg) was made up to lx with

SDS-PAGE sample buffer to a final volume of 25 f..ll using lysis buffer. Heat the

samples in boiling water-bath for 3 min. Spin at maximum speed for lmin. and

keep the tubes on bench till ready for loading. (The samples can be stored at -20°C

and used later.) Also prepare the protein marker as instructed on its product sheet.

Load the samples slowly and smoothly avoiding any spill-over in the adjacent

wells. Run the gel at 80V in 25 mM Trizma® Base (Sigma), 192 mM glycine and

0.1 % SDS (w/v). When the dye front leaves the stacking gel, increase the voltage

to lOOV (Check the buffer level during the run). When the dye front reaches the

bottom most part of the gel, the electrophoresis is terminated. At this point, the gel

can be stained with 0.25 % (w/v) Coomassie Brilliant blue R250 in 20% methanol

and 10% acetic acid and destained (see Recipes Section 1!.13.2) or immediately

processed for Western blot.

Reagents for Preparation of Whole Cell Extract

Lysis buffer (final Concentrations) 40mM HEPES-NaOH, 350mM NaCl, 0.1%

Tween 20 and 10% Glycerol

Protease inhibitor cocktail (final Concentrations): 1 mM PMSF, 2.5 f.!g/ml

Aprotinin, 2 t-tg/mlleupeptin, 2 f.!g/ml pepstatin, 10 f.!g/ml TPCK, 10 t-tg/ml TLCK,

2.0 mM Benzamidine, 1.0 mM DTT.

Protease inhibitors are added to the required volume of extraction buffer just before

use. The stocks are stored as aliquots at -80°C.

36

Western Blotting and Immunodetection

At the end of the electrophoresis, the proteins are transferred to either nitrocellulose

membrane (Hybond ECL, GE Healthcare) or to PVDF membrane (Hybond-P, GE

Healthcare) as suggested by GE Healthcare. The transfer was carried out in 25 mM

Trizma® Base (Sigma), 192 mM glycine, 20% methanol at 30V for 3-4 hours. (The

time of transfer can be standardized for maximum transfer efficiency.) Note the

decrease in current during the transfer. After the transfer is over, the membrane is

removed and stained for 5 min with Ponceau S and destained. The gel can also be

checked for the extent of transfer by staining with coomassie dye. The positions of the

protein marker bands are marked on the blot and the stained blot is scanned and image

recorded. The membrane is blocked in freshly prepared blocking solution (5% milk

powder (BD-Difco) in PBS-T) at RT for -4 hours or keep it overnight at 4°C. The

primary antibody vial is spun for one minute at maximum speed. Make appropriate

dilution of the primary antibody in fresh blocking solution. Incubate the membrane

with primary antibody for one hour at RT with gentle movement on rocker. (The

membrane should be completely covered by the antibody solution). Wash the I

membrane with 1 x PBS-T in three steps. The volume of washing solution has to be

chosen depending on the use of Hybond-P membrane or the Hybond-ECL membrane.

Wash 1- 15ml, 5 min; Wash 2- 25ml, 15 min; Wash 3- 25ml, 15 min. The secondary

antibody HRP-labeled secondary antibody is diluted 1:20,000 in fresh blocking

solution. Incubate the membrane with secondary antibody for one hour at RT with

gentle movement on rocker. Repeat the washing steps as above. The bound antibody is

detected with ECL-Plus Western blot detection system (GE Healthcare) and quickly

exposed to Kodak X-Omat XAR2 X-ray film. The film was developed using Kodak

processing chemicals in a dark room. Multiple exposures were taken for each blot.

37

The film was scanned and images recorded and edited using Adobe Photoshop and

assembled in Adobe Illustrator software.

11.8. Protocol for Immunoprecipitation

All centrifugation steps with Protein G-Sepharose beads should be carried out at 4000

rpm for 2 minutes in a microfuge. After spin, the tubes are placed on the tube-rack and

allowed to sit straight for half a minute. This allows the beads to settle down well.

1. Take 600Jlg protein extract and mix with 2.5Jll of the rabbit anti-CAP2 polyclonal

antibody or preimmune sera and incubate on ice for 1 h.

2. Using a wide-bore micropipette tip (ends to be cut-off straight with a scalpel blade),

pipette out 10J..1.l ofProtein G-Sepharose (Amersham Biosciences, Cat #17-0618-01)

beads in a 1.7 ml microcentrifuge tube (Axygen) for each reaction. Caution:

Remember to completely resuspend Protein G-Sepharose beads before removing an

aliquot.

3. Equilibrate the beads in extraction buffer containing protease inhibitors.

4. Add 5J..1.l of pre-equilibrated Protein G-Sepharose beads to each reaction and

incubate for 1h on a nutator in the cold room at 4°C.

5. Collect the beads by spinning at 4000 rpm for 2 min in cold room. Remove the

supernatant and wash the beads coated with antigen-antibody complex with buffer

three times. An aliquot of the supernatant can also be saved for analysis ..

6. Elute out the immunoprecipitated protein by boiling in SDS sample loading buffer

and proceed for western blot as mentioned above.

38

11.9. Protocol for Total RNA Isolation

Work with gloved hands all the time. All reagents should be prepared fresh and all reagents, glassware

and plasticware should be meant only for RNA work. Lesser the time delay between cell lysis and RNA

precipitation, better the quality and yield of RNA.

1. Resuspend 5-10 OD6oo of cells in 0.5 ml AE buffer (50 mM Sodium acetate, 10 mM

EDTA pH 5.0). Transfer the cell suspension into a 2 ml microfuge tube from

Axygen. Further, add 33.4 J..Ll of 25% SDS and 0.5ml of hot phenol (equilibrated

with AE buffer and prewarmed to 65°C). Transfer tubes immediately to 65°C

water-bath. Incubate for 10 min with intermittent vortexing every 2 min.

2. Bring tubes to RT by keeping on ice for 2-3 minutes (Prolonged cooling may

precipitate out SDS).

3. Spin down at maximum speed for 10-12 min at RT. Two layers can be seen.

Transfer the upper aqueous layer into a fresh 2 ml microfuge tube (Axygen)

containing 0.5ml of chloroform. Mix the content by inversion till immersion forms.

Do not vortex to mix.

4. Spin down at maximum speed for 10-12 min at RT. Collect upper aqueous phase in

fresh tube. Add 1/10th volume 3 M sodium acetate (pH 5.3) and equal volume of

isopropanol. Mix by inversion.

5. Spin down at at maximum speed for 40-50 min at 4°C. Decant supernatant. Wash

RNA pellet with chilled 70% ethanol. Spin down at maximum speed for 15 min at

4°C. Decant supernatant. Give a short spin and aspirate out remaining ethanol.

6. Let pellet dry at RT for 5-10 minutes. Resuspend pellet in 25-50 J..Ll DEPC-treated

autoclaved. Keep on ice and mix by tapping.

7. Determine the yield and purity by spectrophotometric estimation of RNA

concentration (Absorbance at 260 nm and ratio of Absorbance at 260 nm to that at

280 nm) using NanoDrop spectrophotometer. Analyze the quality of RNA

39

preparation on 1.2% 0.5X TAE-agarose gel. Make aliquots of RNA solution and

store at -80°C. A void repeated freeze-thaw cycles.

11.10. RT PCR analysis

DNase treatment: Preparation of RNA samples prior to reverse transcription PCR

The RNA sample was treated with DNase I to get rid of any genomic DNA

contamination. The reaction is set up as follows: 0.5J.tg Total RNA, 0.5J.tl lOx DNasei

Reaction buffer, 0.25J.tl DNasei, Amp.grade (lU/J.tl; Invitrogen Cat. No. 18068-015)

and add RNase-free water to bring volume to 5J.tl. Incubate the reaction at RT for

exactly 15 min. Add 0.5J.tl of25mM EDTA and then heat inactivate the DNase at 65°C

for 10 min. RNA sample is now ready for RT-PCR.

End-product RT-PCR analysis:

(i) 1st strand eDNA synthesis:

Components Stock Final 1 Ox RT Buffer lOx lx 25xdNTP mix 25x 1 X

1 Ox Random Primer lOx lx DNase-treated RNA 100 ng/J.tl lOOng MultiScribe Reverse

(50U/J.tl) 2.5U Transcriptase RN ase free water - Bring to

7.5J.tl

The -RT/+RT reactions are carried out in thermal cycler with the following

conditions 25°C, 10 minutes followed by 37 °C for 120 minutes and 85 °C for 5

seconds 4°C, hold.

(ii) PCR amplification of eDNA:

The reaction mixture included 1 x Taq buffer with 20 mM (NH4)2S04, 2 mM

MgClz, 0.2 mM each dNTP, 0.4 J..LM each primer, 0.35 U Taq DNA polymerase,

1 J.tl of 10 fold eDNA dilution in a total volume of 10 J.tl. The PCR conditions were

40

as follows: 94°C, 2 min (Step 1), 94°C, 15 sec (Step 2), 58°C, 30sec (Step 3), 72°C,

45 sec (Step 4), 72°C, 10 min (Step 5). Steps 2-4 were repeated for 30 cycles.

11.11. Bacterial Expression of Gat9p and Gat9ACp

The recombinant plasmids pHP97 and pHP 104 were transformed into E. coli BL21

(DE3) cells and grown in LB media containing 25 jlg of kanamycin/ml at 37°C to an

OD6oo/ml of 0.5. Expression was induced by the addition of 0.1 mM isopropyl-B-D­

thiogalactopyranoside (IPTG) for 3 h at 37°C. The cells were then harvested,

resuspended in binding buffer (20mM Sodium phosphate pH 7.4, 500 mM NaCl, 20

mM Imidazole) and lysed by sonication.

Both the full length and truncated recombinant Gat9 proteins were found to be

present in the insoluble fraction. To resolubilize the full length recombinant protein, the

insoluble fraction was solubilized in the Binding buffer containing 8M Urea and 1 mM

~-ME. The resolubilized recombinant Gat9-6xHis protein was gradually diluted to

reduce the urea concentration to 4M and loaded onto a His-Trap Affinity column (GE

Healthcare) pre-equilibrated with the Binding buffer containing 4M Urea, 1 mM ~-ME.

The column was then washed with the pre-equilibrated buffer followed by a linear

gradient of 4.0 to 0.0 M Urea for on-column refolding of the protein. However, in spite

of several attempts, the protein precipitated on the column therefore making it difficult

to elute. We then tried to purify the truncated version of the recombinant protein using

the same strategy but unsuccessful. Since the urea solubilized Gat9~C protein in itself

was at least >95% pure as judged by SDS-PAGE (Fig. II. 1.), we decided to cut out the

protein band of interest from a preparative SDS-PAGE gel and use the macerated gel

pieces directly for immunization. This protein preparation was used to raise polyclonal

41

Gat9p Biorad Marker

lx 2x 4x 8x 16x lx 2x 4x

1 2 3 4 5 6 7 8

Fig. 11.1. SDS-PAGE analysis to quantify the recombinant Gat9p.

About 0.25 ml of the urea solubilized Gat9 protein and its 2-fold serial dilutions

(lanes 1-5) were resolved on an SDS-PAGE gel along with 0.25 ml of the BioRad

low range protein marker (2.0mg/ml/band) and its 2-fold dilutions (lanes 6-8). The

gel was stained with Coomassie Brilliant Blue and the specific protein bands

quantitated by comparing their intensities with those of the marker bands.

l

antibodies in BALB/c mice as per established protocols (77). The antibody could detect

both recombinant Gat9p-6xHis and native Gat9p in C. albicans cell extracts.

11.12. Gat9p Antibodies

Bleeds were obtained after 2nd and subsequent boosts, sera collected and the antisera

was used at 1 :200 dilution to probe C. albicans extracts and appropriate recombinant

purified proteins as controls. Serial dilutions of the recombinant proteins (50 J.lg to 0.4

J.lg, 5-fold dilutions) were spotted onto PVDF membranes and the dot blots were also

probed simultaneously. Blots were developed as described in section II. 7. The

antibodies could detect recombinant as well as native Gat9p from cell extracts but also

produced some non specific bands possibly due to cross reaction with some other

C. albicans proteins.

11.12. Recipes of Culture Media, Common Reagents and Buffers

11.12.1. Preparation of growth media

BactoTryptone, Bacto Yeast extract, BactoPeptone and BactoAgar were obtained from Difco laboratories; other chemicals were obtained from Sigma, Merck or Qualigens. Components obtained from specific source will be mentioned otherwise.

YPD IYPM medium

Composition

Yeast extract Peptone Water

200ml

2.0 g 4.0 g Make upto 190 ml

Dissolve the components in water with constant stirring and mild heating. Water

volume at this step is total medium volume minus glucose and maltose volume that is

added after autoclaving. Sterilize the medium by autoclaving for 20 min. Liquid media

is prepared in reagent bottle and stored at RT. Glucose or maltose (stock concentration

42

is 40%) is added to a final concentration of 2% just before use. For preparation of solid

plate media, add 4 g of agar before autoclaving. The glucose or maltose is added after

autoclaving.

SD (Synthetic Dextrose) or SM (Synthetic Maltose) media plates.

Composition/Litre

Yeast Nitrogen Base* (YNB) 1. 7 g Ammonium sulphate 5 g Agar 20g Water 800 ml

* YNB without ammonium sulphate and amino acids

Dissolve the components in water with constant stirring and mild heating. Water

volume at this step is total medium volume minus glucose and maltose volume that is

added after autoclaving. Sterilize the medium by autoclaving for 20 min. Liquid media

is prepared in reagent bottle and stored at RT. Glucose (stock concentration is 40%) is

added to a final concentration of 2% just before use. For preparation of solid plate

media, add 20 g of agar before autoclaving. Glucose/maltose is added after autoclaving.

Luria-broth (LB) medium, 250 m1

Composition

Yeast extract 1.25 g Tryptone 2.5 g NaCl 2.5 g Water make up to 250 ml

Dissolve the components in water with constant stirring and mild heating. Water

volume is adjusted after adjusting the pH 7.0 with 10 N NaOH. Sterilize the medium by

autoclaving for 20 min and store the medium at RT.

For preparation of LB plates containing ampicillin (LB-Amp), add 5 g of agar after

adjusting the pH and before autoclaving. After autoclaving, place the media on the

stirrer so that temperature lowers down to -60 °C, add ampicillin from the sterile stock

43

solution of 25 mg/ml to a final concentration of 75 J.l.g/ml. Mix it well on stirrer and

pour the plates.

Spider Media: 1 OOml

1.0 g Nutrient Broth, 1.0 g Mannitol, 0.2 g K2HP04 and Water to make up to 100 ml.

Dissolve the components in water with constant stirring. Sterilize the medium by

autoclaving for 20 min and store the medium at RT. For preparation of agar plate, add

2.0 gm of Bacto-agar before autoclaving.

44

11.12.2. Recipes for making common buffers and reagents

REAGENTS

TAE,pH8

Trisbase

Glacial Acetic Acid

EDTA

Tri-sodium pH8

Di-sodium pH8

TE,pH8

EDTA,

EDTA,

Tris-HCl, pH 8

EDTA,pH8

Congo Red

Caffeine

LiCl

CaC}z

Tris-HCl, pH 8.0

TBE

Trizma® base

Boric acid

EDTA

SDS

TBS,pH7.6

Trizma® base

Sodium Chloride

Cone. HCl

STOCK

20X

0.5M

0.5M

0.5M

lX

1M

O.SM

1M

25mg/ml

15mg/ml or77mM

1M

2.5M

1M

sx

0.5M

10%

lOX

AMOUNT

100ml

9.68 g

2.28 ml

4.0ml

PREPARATION

Dissolve the components with mild heating on magnetic stirrer and make up the volume. Autoclave and store at 4°C.

60 ml; 10.75 Dissolve in 30 ml water; stir vigorously. Adjust the pH g tri-sodium by NaOH. Make up the volume. Autoclave and store at EDTA 4°C.

60 ml; 11.17 g di-sodium EDTA

50ml

0.5 ml

0.1 ml

lOml

10ml

10ml

20ml

10ml

Dissolve in 40 ml water, stir vigorously. Adjust the pH by adding -3 ml of 10 N NaOH. Check pH after every 1 ml NaOH addition. Stir vigorously. EDTA will go into solution at pH 8. When solution becomes clear, make up the volume. Autoclave and store at 4°C.

No need to autoclave when using sterile components.

Dissolve 2g in 7ml of water. Adjust volume to lOml and autoclave to sterilize. Dissolve 250mg into water. Filter sterilize by passing through 0.221Jlll filters and store at 4 degree in Dissolve 150mg into water. Filter sterilize by passing through 0.22Jlm filters and store at 4 degree in

Dissolve 8.478g into -12ml water. Adjust volume to 20ml. Filter sterilize by passing through 0.22Jl filters and store at 4 degree in aliquots. Dissolve 3.675g in -7ml. Adjust volume to lOml. Filter sterilize by passing through 0.22J.1m filters and

50 ml; 6.05 g Dissolve in 40 ml water; adjust the pH to 8.0 by cone. Trizma® HCl and make up the volume. Autoclave and store at base 4°C.

500ml

27 g

13.75 g

10ml

60 ml; 6 g SDS powder

120ml

2.9 g

9.6 g

-Q.4ml

Dissolve Tris and Boric acid in water; add EDT A from stock solution and make up the volume. Autoclave and store at 4°C.

Dissolve gradually in 30 ml water and make up the volume. Do not autoclave. It can be filter sterilized and store at RT.

Dissolve Tris and boric acid in water. Check pH; adjust pH with HCl and make up the volume. Autoclave and store at 4°C.

45

DYE STOCK AMOUNT PREPARATION

DNA loading buffer 6X 25 ml

Sucrose 40% 10 g

Bromophenol Blue Weigh the components and suspend in autoclaved

0.25% 62.5 mg MQ water. Add sucrose first and dissolve it

1 M Tris-HCl, pH 8 lOmM 250 J.1L completely. Add BPB at the last. Nutate for sufficient time. Store at 4°C.

0.5 M EDTA, pH 8 lOmM 500 J.1L

SDS-PAGE Protein Loading buffer 2ml

Tris-HCl, pH 6.8 2M 0.375 ml Add 240 mg of SDS to a 2.0 ml vial with pre-warmed 0.6 ml USB glycerol; add 375 J.ll ofTris-Cl

Glycerol 100% 1.2ml buffer into it. Vortex vigorously and keep in water-

BPB bath set at 55°C; repeat process. Add remaining glycerol; mix well and make up volume to 2 ml.

SDS 240mg Add BPB and mix on nutator.

DNA Denaturing lml PAGE-loading buffer

Formamide 100% 0.9 ml The volume of the dye is adjusted so that the final formamide concentration is 50%; in 12 J.ll loading

EDTA,pH8 0.5M 2 J.ll prep of the sample, 6.67 J.ll of dye is added. Very

BPB

Reagent Stock Preparation

Weigh out 100 mg in a 15 ml falcon tube. Add 4 ml of water and Ampicillin, 4ml 25mglml dissolve by putting on nutator. Filter sterilize using 0.22J.1

membrane. Make aliquots of0.5 ml and store at -20°C.

Weigh out 50 mg in a 15 ml falcon tube. Add 5 ml of water and Kanamycin, 5ml lOmglml dissolve by putting on nutator. Filter sterilize using 0.22J.l

membrane. Make aliquots of0.5 ml and store at -20°C.

Weigh out 68 mg in a 2 ml eppendorff tube. Add 2 ml of ethanol Chloramphenicol, 2ml 34mg!ml and dissolve by putting on nutator. Make aliquots of 0.5 ml and

store at -20°C.

Weigh out 10 mg in a 15 ml falcon tube. Add 10 ml of water and Fluconazole, 1 Oml lmglml dissolve by putting on nutator. Filter sterilize using 0.22J.l

membrane. Make aliquots of0.5 ml and store at -20°C.

Weigh out 25 mg in a 15 ml falcon tube. Add 5 ml of 100% Mycophenolic Acid, 5ml 5mglml ethanol and dissolve by putting on nutator. Make aliquots of 0.5

ml and store at 4°C. Weigh out 17 .5mg in a 15 ml falcon tube. Add 5 ml of water and

Calcofluor white, 5ml 3.5mglml dissolve by putting on nutator, add drops of ION NaOH till the solution becomes clear.Filter sterilize using 0.22J.1 membrane. Make aliquots of0.5 ml and store at -20°C. Weigh out 50 mg in a 15 ml falcon tube. Add 5 ml ofDMSO and

Caspofungin, 5ml lOmglml dissolve by putting on nutator. Filter sterilize using 0.22J.1 membrane. Make aliquots of0.5 ml and store at -20°C.

Weigh out 25 mg in a 15 ml falcon tube. Add 5 ml ofDMSO and Brefeldin, 5ml 5mglml dissolve by putting on nutator. Filter sterilize using 0.22J.1

membrane. Make aliquots of0.5 ml and store at -20°C. Weigh 10 mg and dissolve in 850 J.ll of 10 mM sodium acetate (pH 5.2). Heat to 100 °C for 15 min to inactivate any

Ribonuclease A 10 mglml contaminating DNase. Allow it to cool slowly to RT. Adjust the pH to >7.6-8.0 using 1 M Tris-Cl, pH 8.0. Make up the volume to 1 ml with 10 mM sodium acetate (pH 5.2). Dispense into aliquots and store at -20 °C.

46