Appendix 11

I APPENDIX 1: MEDIA AND SOLUTIONS

All % shown are on a W/V basis unless mentioned otherwise. All solutions and media

were made in Milli-Q and Milli-RO water respectively. All solutions and media were

sterilized by autoclaving at 15 lb/ sq inch for 15 minutes or filter sterilized by passing

through a 0.221lM Millipore filter. Media used were from Invitrogen or Difco.

Chemicals used were of analytical grade (Qualigens or Merck) or molecular biology

grade mostly from Sigma, Invitrogen or USB. Restriction enzymes and DNA

modifying enzymes were obtained from NEB, MBI-Fermentas, Roche, Promega,

Amersham or Perkin Elmer. Buffers provided with the enzymes were used.

ALl. Bacterial Media:

1. Luria Bertani (LB) Medium (Invitrogen): 1 % Tryptone, 1 % NaCl, 0.5% Yeast Extract

2. LB agar:

3. Terrific Broth:

.. .

4. 2 X L Broth:

LB with 1.5% Agar (Invitrogen).

Antibiotics were added to the media, after

autoclaving, and cooling to <50°C, at

indicated concentrations. Ampicillin was

added to the concentration of 50Ilg/ml, to

liquid medium and 75Ilg/ml, to Agar

medium. Streptomycin was added at the

concentration of 10llg/ml to liquid media

and 15 Ilg/ml to Agar medium.

1.2% Bacto-tryptone (Difco), 2.4% Yeast

extract (Invitrogen), 0.4% (v/v) glycerol.

Supplemented with one tenth volume lOX

Phosphate Buffer (2.31g K2HP04, 12.54 g

KH2P04 in 100ml H20).

2% Bactotryptone, 1 % Yeast Extract,

0.1 % NaCl, 0.2% glucose.

86

5. SOC:

6. GYT:

Appendix 1

0.5% Yeast Extract, 2% Tryptone, 2.5mM

KCI, 10mM MgCb, 10mM MgS04,

20mM dextrose. Filter sterilized.

Glycerol 10%· (v/v) , Yeast Extract

0.125%, Tryptone 0.25%.

Al.2. Media for growth, maintenance, two-hybrid and morphogenetic studies

of Candida albicans:

1. YPD:

2. YPDAgar:

3. SD:

4. SD Agar:

5. SN:

6. SN Agar:

7. 5-FOA Plate:

8. GPK:

1 % Yeast extract, 2% Peptone, 2%

Dextrose

YPD with 2% agar

0.67% Yeast nitrogen base without amino­

acids (YNB wlo aa), 2% dextrose,

supplemented with Uridine at the

concentration of 25 /-lg/ml, when required.

SD with 2.0% agar.

0.67% Yeast nitrogen base without amino­

acids (YNB wlo aa); 2% G1cNAc.

SN with 2% agar.

0.67% YNB wlo aa, 2% Dextrose,

1mg/ml

5-Fluoroorotic Acid (filter sterilized and

added when media is <50°C), 25/-lglm1

Uri dine and 2% Agar.

0.5% Glucose, 0.5% Peptone and 0.3%

K2HP04

87

9. NPK:

10. Spider (pH 7.2):

11. SLAD:

12. 2.5mM GlcNAc in salt base:

13.20% Serum:

14. SCAA

15.10X Dropout solution:

Appendix 1

0.5% GlcNAc, 0.5% Peptone and 0.3%

K2HP04

1 % Nutrient Broth, 1 % Mannitol, 0.2%

K2HP04, 1.35% Bactoagar for plates.

Adopted from Liu et aI., 1994.

0.17% YNB w/o aa and (NH4)2S04, 2%

Dextrose, 2 % Bactoagar (washed several

times with autoc1aved water) and 50llM

filter sterilized (NH4)2S04, added after

autoc1aving. Adopted from Gimenco et aI.

1992.

0.45% NaCl and 0.335% YNB w/o aa.

Adopted from Delbruck and Ernst, 1993.

20% Bovine Calf Serum in YPD.

0.67% YNB (w/o aminoacids); 2%

Casamino acids (Difco ). Adopted from

Leuker et aI., 1997.

L-Isoleucine­

L-Valine-

300mg/ml

1500mg/ml

L-Adenine hemisulphate-200mg/ml , L-Arginine-HCl- 200mg/ml

L-Histidine-HCl monohydrate-200mg/ml

L-Leucine- 1000mg/ml

L-Lysine HCl- 300mg/ml

L-Methionine- 200mg/ml

L-Phenylalanine- 500mg/ml

L-Threonine- 2000mg/ml

L-Tryptophan- 200mg/ml

L-Tyrosine- 300mg/ml

88

Appendix 1

L-Uracil- 200mg/ml

For Dropout Selection Medium

appropriate

components were omitted from the lOX

dropout solution. The solution was

autoclaved and stored at 4°C.

A1.3. Reagents and Buffers used for bacterial transformation, plasmid and

genomic DNA isolation, and molecular cloning:

I. Trituration Buffer: 100mM CaCh, 70mM MgCh, 40mM

NaOAc (pH 5.5).

2. X-Gal:

3. IPTG:

4. Solution I:

5. Solution II:

6. Solution III:

7. TE (pH 8.0):

8. TE (pH 7.6):

9. 3M NaOAc (pH 5.2):

20 mg/ml X-gal, in DMF. Stored at -20°C

in dark.

200 mg/ml IPTG, in water, filter sterilized

and stored at -20°C.

25mM Tris-HCI, pH 8.0; IOmM

EDT A; 50mM Dextrose.

1 % SDS, O.2N NaOH.

Potassium acetate, 3M with respect to

Potassium and 5M with respect to acetate,

as per Sambrook et aI., 1989.

10 mM Tris-HCI (pH 8.0), ImM EDTA

. (pH 8.0).

IOmM Tris-HCI (pH 7.6), ImM EDTA

(pH 8.0).

40.81gm of sodium acetate trihydrate was

dissolved in 80ml water. pH was set to 5.2

89

11. Phenol, Qualigens:

12. TE Saturated phenol:

13. Phenol: Chloroform: IAA:

14. RNase A:

IS. Proteinase K:

16. NaCISM:

17. Acrylamide gel elution buffer:

18. Lysozyme:

19. 2X PEGlNaCI:

Appendix 1

with glacial acetic acid. The volume was

adjusted to 1 OOml.Sterilized by autoclaving

38.Sg of ammonium acetate was dissolved

in and the volume was adjusted to 100

ml and autoclaved.

Redistilled at 180°C and stored frozen at

minus 20°C in small parts.

Prepared according to Ausubel et al (1994)

2S parts of TE saturated phenol, 24 parts

of chloroform and 1 part of

Isoamylalcohol.

10 mg/ml. Stock was prepared as

described in Sambrook et aI., (1989).

20mg/ml in water.

29.22gm ofNaCI was dissolved in 80ml

water and the volume was adjusted to

100ml. Sterilized by autoclaving.

O.SM ammonium acetate, 10mM EDTA

(pH 8.0), 0.1 % SDS (optional).

10mg/ml in lOmM TrisCI pH 8.0.

13% (w/v) PEG 8000 and 1.6M NaCI.

Al.4. Reagents and Buffers used for Agarose Gel Electrophoresis and

Polyacrylamide Gel Electrophoresis:

1. SOX TAE: 242g Tris base, S7.1ml Glacial acetic acid

and 100ml O.SM EDTA, pH 8.0 for 1 litre.

90

2. Ethidium Bromide:

3. EndoR (6X):

4.40% Acrylamide:

5.25%APS:

6. lOX TBE:

Appendix 1

10mg/ml in water.

30% Ficoll 400, 60mM EDTA pH 8.0,

0.6% SDS and 0.06% bromophenol blue.

29: 1 Acrylamide and Bis-acrylamide was

prepared and deionised as recommended

in Sambrook et aI., 1989.

Prepared freshly by dissolved at 250mglml

in water.

54g Tris base, 27.5g boric acid and 20ml

0.5M EDTA was added for llitre soln.

At.S. Reagents and Buffers used for C.albicans transformation and Genomic DNA

isolation:

1. LATE buffer:

2. PLATE buffer:

3. Sheared Salmon Sperm DNA:

4. Breaking Buffer:

Lithium acetate 100mM, Tris-HCI pH 7.5

10mM, EDTA ImM.

40% PEG 3350 in LATE buffer.

10mg/ml in lXTE pH 8.0, sonicated and

extracted with phenol: chloroform: IAA

twice and once with chloroform: IAA.

Precipitated with ethanol, washed and

resuspended. Prepared as per Sambrook et

aI., (1989).

10mM Tris-HCI, pH 8.0, 1 % SDS, ImM

EDTA, 2% Triton-X-I00 and 100mM

NaCI.

A1.6. Buffers used for Southern Blot analysis and Hybridization:

1. 20X SSC: 175.3 g NaCI and 88.2 g trisodium citrate

were dissolved in 800 ml MQ water. The

pH was adjusted to 7.0 by adding 20001

91

2. Depurination Solution:

3. Denaturation Solution:

4. Neutralizing Solution:

S. Post Transfer Denaturation Solution:

6. Post Transfer Neutralization Solution:

Appendix I

of SO% HCI, volume was made up to I

litre and sterilized by autoclaving.

O.2SN HCI.

0.6M NaCI and O.4N NaOH.

I.SM NaCI, O.SM Tris HCI pH 7.S

O.4NNaOH

0.2 M Tris-HCI pH7.S and IXSSC.

A1.7. Reagents and Buffers used for RNA isolation and Northern Blotting:

1. TES: 10mM TrisCI pH 7.S, lOmM EDTA, O.S%

SDS, prepared in DEPC water.

2. Phenol:

3. 2M NaOAc (pH 4.2):

4. Loading buffer:

S. lOX MOPS:

Water saturated.

27.2lgm was dissolved in 80ml water, pH

set to 4.2 with glacial acetic acid, and

volume adjusted to 100mi. Autoclaved and

stored at R T.

SO% glycerol, ImM EDTA, 0.002%

bromophenol blue and 0.002% xylene

cyanol

O.2M MOPS (pH 7.0), 20mM sodium

acetate, 10mM EDTA. Dissolve 41.8g of

(3-[N -morpholino ]propanesulfonic acid)

in 700ml of sterile DEPC-treated water,

adjust pH to 7.0 with 2N NaOH. Add

20ml of DEPC-treated 1M sodium acetate

and 20ml of DEPC-treated O.SM EDT A

(pH 8.0). Adjust the volume of the

solution to 1 liter with DEPC-treated

92

6. Methylene blue solution:

Appendix I

water. Sterilize the solution by filtration

through a 0.45-/lm Millipore filter, and

store it at room temperature protected

from light. The buffer yellows with age if

it is exposed to light or is autoc1aved.

0.02% methylene blue in 0.3M Sodium

Acetate.

A1.8. Buffers and Solutions used for Hybridization of DNA and RNA blots:

1. Prehybridization Buffer: 1 % SDS, 2X SSC, 10% Dextran Sulphate

and 50% deionized Formamide.

2. Posthybridization Wash Solution I: 2XSSC

3. Posthybridization Wash Solution II: 2X SSC and 1 % SDS

4. Posthybridization Wash Solution III: 0.2X SSC and 1 % SDS

A1.9. Buffers and solution for Western Blotting:

1. 4X Tris-CI pH6.8:

2. 8X Tris-CI pH 8.8:

3. IX SDS gel loading buffer:

4. Staining solution:

6.05gms of Tris base was dissolved in 40ml

MQ, pH adjusted with IN HCI and volume

Made to 100 ml. Filtered through

Whatmann filter.

182 gms. ofTris base was dissolved in 300 ml

MQ. The pH was adjusted to 8.8 with IN HCI

and the volume was made up to 500 ml and

filtered.

50mM Tris-HCI, 2%SDS, 0.1 %Bromophenol

Blue, 10% Glycerol, 100mM DTT.

Methanol 40%, Acetic acid 10%,MQ 50%,

Coomassie 0.2%.

93

5. Destaining solution:

6. Ponceau S satin:

7.10XTBS:

8. TBST:

9. Transfer buffer:

Appendix 1

Methanol 40%, Acetic acid 10%, MQ 50%.

0.5% Ponceau S in 1 % acetic acid.

100mM Tris pH 8.0, 150 mM NaCl.

lXTBS with 0.1% Tween 20.

20% methanol, 24 mM Tris base, 194 mM glycine.

94

Appendix 2

I APPENDIX 2: COMMONLY USED TECHNIQUES

A2.1 In vitro DNA manipulation:

A2.1.1. Restriction Analysis: The restriction analyses of the DNA was done after digestion according to the

manufacturer's recommendations with regard to the buffers used, incubation time and

temperature and amount of enzyme per IJg of DNA used, which were strictly adhered

to. The digests were then mixed with loading buffer EndoR to a final concentration of

IX, and loaded on to a gel. After electrophoresis, the gels were phptographed using a

gel documentation system Alfa Imager, or a Polaroid camera. For routine gels, a trace

of the gel was made on a polythene sheet. The size of the fragments was estimated by

measuring the relative mobility of the bands in comparison to markers of known

molecular size, ego lkb or 100bp DNA ladders (Gibco BRL) or A genomic DNA

digested with HindIIl, run in a lane alongside. The mobility was converted to size by

using a program, based on the method of estimation of size of DNA by Southern.

Depending upon the size of the fragment, the position of the restriction site on the DNA

was calculated.

A2.1.2. peR Amplification and Optimization

PCR was carried out in an MJ Research PTC I 00 therrnocycler, with a heated bonnet or

in PTC200 for programmes that needed gradient annealing temperatures.

A2.1.2.1. Amplification

Typically the amplification reactions were done for ~30 cycles, with specific alterations

arrived at, empirically to optimize yield. The program was as follows,

94°C denaturation for 2 minutes only in the first cycle

Optimum annealing temperature Ta OPT for 30 seconds, for all cycles

94°C denaturation for 1 minute for all cycles

72°C extension for I minute for all cycles

Cycling for ~30 times

noc for 10 minutes to fill the incomplete extension products,

4°C for a minimum of 10 minutes.

95

Appendix 2

The extension temperature was set at 68 DC while working with the enzyme Accuprime

Pix (Invitrogen).

A2.1.2.2. Optimizations

1. Optimization of Annealing Temperature:

The annealing temperature was calculated using the following parameters according to

Rychlik et al., 1990.

T a Primer=O. 3 T m Primer + 0.7 T m Product

TmProduct = 0.41(%G+C) +16.6 (lOglO [K+]) + (67511) + 81.5 DC,

Where, I defines the length of the product in bp, and [K+] represents the potassium ion

concentration in Ix PCR buffer which is normally provided as 50 mM.

And Tm Primer =2x (A+T) + 4 X (G+C), where A, T, G and C are the number of the

respective nucleotides in the oligonucleotide primer sequence.

The T a OPT was usually taken as the lower of the two T a Primers calculated as above.

The T a thus arrived at, was quite close to the ideal temperature, just a difference of

0.7DC.

2. Optimization ofMgCb:

The Mg ++ ion concentration of the amplification reaction was optimized empirically,

varying the concentration from 1mM to 4mM in increments of 0.5 mM to obtain the

best amplification.

3. Optimization of yield:

Using the above parameters, the template amount, and number of cycles were varied

and an ideal combination was arrived at to maximize the yield.

A2.2. Gel Electrophoresis and Gel Elution of DNA fragments

1. Agarose Gel Electrophoresis:

Agarose gel electrophoresis of DNA was routinely carried out in IX TAE, in the

presence of 0.5J.Jg/ml ethidium bromide (A1.5.2) both in gel and in running buffer. For

most purposes 0.8% gel was used. Depending on the expected size of the band, the gel

strength was varied from 0.7%, for visualizing uncut genomic DNA, to 2.0% for

checking total RNA. Before performing any southern or northern, the integrity of the

nucleic acid was also verified by such methods. For the exact measurement of very

96

Appendix 2

small fragments, ranging from 75 to 500bp of DNA, nondenaturing Polyacrylamide Gel

Electrophoresis (PAGE) was carried out as described later (A2.2.3). All procedures

were followed from Sambrook et aI., 1989, or Ausubel et aI., 1994.

2. Gel Elution of inserts from Agarose Gel:

Large scale digestion of the plasmid containing the DNA of interest was set with

appropriate enzymes to release the insert. Preparatory gel was casted using low melting

agar. The digested DNA was loaded on the gel and run at low voltage till the band of

interest was well separated from the vector backbone. The band was cut out with a new

scalpel. Equal volume of TE saturated phenol was added to it and kept in minus 80°C

for 1 hour. It was then centrifuged at 13,000 rpm at room temperature, and the aqueous

layer was transferred into a new tube. The DNA was extracted once with chloroform:

isoamyl alcohol and precipitated by adding 1/1Oth volume of 3M NaOAc (A1.4.9) and

2.5 vols of chilled ethanol. The precipitate was washed, dried and dissolved in 20111 TE.

Alternatively the DNA was prepared from the gel slice using QIAEX II Gel Extraction

Kit (QIAGEN). PCR products were purified from the gel similarly.

3. Nondenaturing Polyacrylamide Gels:

These were used for the separation and purification of fragments of double-stranded

DNA. 8% gel was prepared for separating fragments which were 60-400bp long. 26.6

ml of 29% Acrylamide and 1 % bisacrylamide stock solution (Al.5.6) were mixed with

52.7ml of water. 20ml of lOX TBE (A1.5.8) and 0.7ml of 10% APS (A1.5.7) were

added to it. Gel apparatus was assembled and gel was poured. Comb was inserted

immediately and the gel was left to polymerize. After the gel had completely

polymerized, the comb was removed carefully and the samples were loaded along with

appropriate markers. The gel was run in IX TBE. When the run was over, the gel was

disassembled and stained in Ethidium bromide.

4. Gel elution of fragments from PAGE:

Crush and soak technique by Maxam and Gilbert (1977) was followed. A

polyacrylamide gel electrophoresis of the DNA sample and markers was carried out as

described previously. The DNA of interest was located by examination of the ethidium

bromide stained gel in long-wavelength (302nm) UV light. A clean sharp scalpel was

97

Appendix 2

used to cut out the segment of gel containing the band of interest, keeping the size of

the polyacrylamide slice as small as possible. The gel piece was then transferred to a

micro centrifuge tube, and crushed against the walls with the help of a disposable

pipette tip. 1-2 vol. ofacrylamide gel elution buffer (A1.4.18) was added to it.

The tube was incubated at 37°C on a rotary platform for 3-4 hours. The sample was

then centrifuged at 4°C at maximum speed for 1 minute. The supernatant was

transferred to a fresh microfuge tube, and an additional 0.5 volume of acrylamide gel

elution buffer was added to the polyacrylamide pellet. It was vortexed briefly, and

centrifuged again. Supernatant was collected and pooled. 2vol of ethanol at 4°C was

added to it, and stored on ice for 30 minutes. The DNA was recovered by centrifuging

at maximum speed for 10 minutes. The pellet was washed with 70% alcohol, dried, and

dissolved in 10).11 of IX TE, pH 8.0.

A2.3. Cloning of gene of interest in vectors

Vectors most commonly used in these studies are pGEMT-easy and pUC19. Enzymatic

manipulations of DNA, like restriction digestion, Klenow end filling,

dephosphorylation by ClAP, ligation, etc. were carried out essentially as described in

Sambrook et aI., 1989 and following the manufacturers' instructions, wherever

applicable. All the preparative digestions for the preparation of inserts and vectors were

generally set up in 20 , .. 11 volume with 10-15 JJg of plasmid DNA. Digests were resolved

on 0.7-1.0% agarose gel and appropriate DNA fragments were cut out from the gel. As

reference, marker DNA ladders were loaded along with the samples. DNA was eluted

from the gel piece either using QIAEX II Gel Extraction Kit (QIAGEN) or by phenol

freeze method. DNA amounts were empirically estimated. Modifications like end

polishing, dephosphorylation, etc. were carried out and recovered DNA was further

estimated. Ligation reactions were set up at vector: insert ratio of 1:4 (in general) in 10

JJl reaction volume containing IX ligase buffer and 3.3 units ofT4 DNA ligase (NEB).

For blunt end ligation, additionally, PEG (MW 8000) was added to a concentration

15% and incubated at 20°C for 16 hours. Sticky end ligation was carried out at 16°C.

Cloning in pGEM-T Easy was carried out as per manufacturers' instructions.

98

A2.4. Transformation of E. coli

A2.4.1. Hanahan Method for preparation of competent cells

1. Preparation of E. coli DHSa Competent Cells

Appendix 2

DH5a was tested for recA as described in Maniatis et al. (1982). Chemically competent

cells were made according to the method described by Hanahan (1991) with some

modifications.

DH5a cells were streaked on fresh LB-agar plate from glycerol stock. A single colony

was inoculated from the freshly streaked plate, in a preculture of IOml of 2XL medium

supplemented with 0.2% glucose (AI.I.4). It was grown at 30°C at 200 rpm shaking

for 16 hours.

The overnight grown preculture was diluted 1: 100 times in fresh 2XL supplemented

with 0.2% glucose and incubated at 30°C with shaking at 200rpm till A600 reached 0.45.

The bacterial culture was then chilled on ice-water bath for 2 hours. The cell culture

was transferred to chilled SM34 tubes and pelleted at 5000 g for 5 min at 4°C in a

chilled rotor. The supernatant was discarded and the pellet was resuspended in half the

culture volume of freshly made trituration buffer (AI.4.I). The cell mix was incubated

on ice-water bath for 45 minutes. Cells were pelleted by centrifugation at 5000 g for 10

minutes at 4°C. Competent cells were then resuspended in trituration buffer in a 1I20th

volume of the starting culture volume. 80% glycerol was added to it to a final

concentration of 15% (v/v).

Cells were ali quoted on ice and immediately frozen at -80°C in microfuge tubes and

stored at the same temperature until further use.

2. Transformation of compet~nt cells

For each transformation, a 100 1-11 aliquot of the competent cells was used. Cells were

thawed on ice. 5-10 1-11 ofligation mix or 1-20ng of plasmid DNA was mixed with I X 1

TE pH 8.0 (A1.4.7) to make a final volume of 50 1-11. 100 1-11 cells was added to it and

incubated for 45 minutes on ice with gentle tapping at regular intervals. The cells were

subjected to heat shock at 37°C in a water bath for 5 minutes and then chilled on ice for

5 minutes. 3.85 ml of2XL broth was added and was allowed to outgrow for 90 minutes

at 37°C at 200 rpm shaking. As a positive control of transformation, lng of intact

pBluescript II vector was used and a negative control was kept without addition of

DNA. Transformation efficiency was determined by counting the number of colonies /

99

Appendix 2

~g of DNA on selection plate. In case of transformation of ligation mix, a vector only

control ligation was also transformed along with vector-insert ligation, to check the

efficiency of dephosphorylation reaction of the vector.

3. Plating of Transformation Mix

40 ~l of20 mg/ml X-gal (Al.3.2) and 4 ~l of200 mg/ml IPTG (Al.3.3) were spread on

25 ml LB-agar plate containing 75~g/ml ampicillin. An aliquot of 200 ~l out of the 4

ml-transformation mix was spread on each plate. The plates were dried in laminar flow

air and then incubated in 37°C incubator, agar side up until the colonies appeared

(16 hours). The plates were then stored at 4°C until blue and white coloured colonies

became distinct.

In the case where blue/ white selection was not present, transformation mixes were

plated on LB ampicillin plates.

A2.4.2. High-efficiency electro-transformation of E.coli

1. Preparation of cells

E.coli strains DH5a was grown overnight in 10ml LB at 37°C with shaking at 200rpm.

1 % inoculum (the entire pre culture) was added to llitre LB and grown at 37°C with

shaking at 200 rpm to an OD6oo of 0.5- 0.6. The cells were chilled on ice water for 30

min. They were centrifuged in a cold rotor at 4,000 rpm for 15 mins. The supernatant

was discarded and the pellet was resuspended in equal volume of 10% ice cold

glycerol. It was centrifuged at 4,000 rpm for 15 mins. The supernatant was discarded

and the cells were resuspended in half the original volume of ice cold 10% glycerol. It

was centrifuged at 4,000 rpm for 15 mins. The supernatant was discarded and the cells

were resuspended in 20 ml of ice-cold 10% glycerol. The suspension was centrifuged at

4,000 rpm at 4°C, for 15 mins. The supernatant was discarded and the cells were finally

resuspended in 2ml of ice-cold GYT (Al.l.7). The cells were ali quoted in 40~1 volume,

snap freezed in liquid Nitrogen and stored in -80 degree for further use.

2. Desalting of ligation mix

1.1 % agarose was prepared and 100mM glucose was added to it. It was poured into a

new microfuge tube with a 0.2 ml tip inserted into it and allowed to solidify. After

solidification the tip was carefully pulled out. This created a small well in the agarose

100

Appendix 2

of approximately 100 III volume. The ligation mix was placed in the well and left

undisturbed for 90 mins. The mix was recovered by carefully pipetting out and kept in a

new tube.

3. Electro-transformation and plating

Electro-transformation was done in Gene Pulser Apparatus (BIO RAD). The cuvette

slide of the apparatus was placed on ice and equilibriated at O°C before the pulse.

Sterile cuvettes (O.2cm) were removed from their pouches and placed on ice.

The frozen cells were thawed at room temperature and then placed on ice, was mixed

with the entire desalted ligation mix and kept on ice for 1 minute. The Gene Pulser

apparatus was set at 2SIlF and 2.S KV. The Pulse Controller was set to 200 ohm. The

mixture of cells and DNA was transferred to a cold electroporation cuvette at the

bottom. The cuvette was placed in the chilled safety chamber slide and the slide was

pushed into the chamber until the cuvette was seated between the contacts at the base of

the chamber. The pulse was given and the time was noted.

The cuvette was removed from the chamber and Iml of SOC (A1.1.6) was quickly

added to it. The cell suspension was transferred to a ISml polypropylene tube and

incubated at 37°C for 1 hour with 200rpm shaking. The transformation mix was plated

on LB plates with appropriate antibiotic, and incubated at 37°C for 16 hours.

A2.5~ Screening and Analysis of Recombinants

A2.5.1. Plasmid isolation

The recombinant colonies (white), from vector and insert plate were picked up and

patched on a fresh LB-agar plate containing ampicillin and simultaneously inoculated

into 1.0 ml TB medium containing SOj.lg/ml ampicillin. The cells in liquid culture were

grown at 37°C in an incubator shaker at 200 rpm for 18-20 hours and the plate was

incubated in 37°C incubator until the patched colonies appeared.

The cultures were processed to isolate plasmid DNA by the mini prep protocol and

analyzed by restriction digestion.

1. Small Scale Plasmid DNA Isolation

Small scale plasmid DNA isolation was carried out by modified alkaline lysis method

of Sambrook. (1989). Cells were pelleted at 12,000 g for 20 seconds at room

101

Appendix 2

temperature (RT). The supernatants containing the media were removed by aspiration,

leaving the bacterial pellet as dryas possible. The pellets were resuspended completely

by vortexing in 50 III of Solution I, TEG (A 1.4.4). 100 1-11 of freshly prepared Solution

II (A1.4.5) was added to them and the tubes were inverted few times to mix well. They

were incubated at RT for 5 minutes till a visible cell lysis occurred and the liquids

became transparent.

75 1-11 Solution III (A1.4.6) was added to the cell lysates and mixed well. They were

incubated on ice for 10 minutes to allow the precipitation of cell debris. They were then

centrifuged at 12,000 g for 7 minutes at RT. The pellets of bacterial debris were

removed using sterile toothpicks.

The plasmids were precipitated by the addition of equal volumes of isopropanol to the

supernatants. They were mixed by inversion and incubated at RT for 15-30 minutes.

The plasmid pellets were recovered by centrifugation at 12,000 g for 5 minutes at RT.

The supernatants were removed by gentle aspiration; pellets were washed once with

70% ethanol and dried under vacuum. The pellets were dissolved in 20 1-11 TE, pH 8.0.

2 1-11 of each sample were separated on a 0.8 percent agarose gel along with the marker

plasmid which was the uncut vector used for ligation. Recombinants were selected

based on their slower migration in comparison to the vector. The putative positive

clones were verified by restriction digestion with suitable enzymes.

2. Medium Scale Plasmid DNA Isolation

A confirmed positive clone was inoculated for Midi-preparation of plasmid DNA in

50ml TB. Alkaline lysis method was done as described by Sambrook et al. (1989) with

modifications as described below.

A single colony containing the plasmid from LB-agar plate supplemented with

Ampicillin (75 I-Ig/ml) was inoculated in 50 ml TB medium supplemented with the

same antibiotic. The cells were grown overnight (16-18 hours) at 200 rpm in a 37°C

incubator shaker. The culture was harvested by centrifugation at 8000 g at 4°C for 5

minutes. The cells were resuspended in freshly made 5 ml of Solution I (A1.4.4) by

vortexing. After thorough suspension, lysozyme (A1.4.19) was added to a final

concentration of Img/ml and incubated at RT for 10 minutes.

10 ml of freshly prepared solution II (Al.4.5) was added to the suspension and was

mixed gently by inversion and incubated at RT for 10 minutes. 7.5 ml of 3M NaOAc,

102

Appendix 2

pH 5.2 (Soln. III, A1.4.6) was added to it and mixed thoroughly by inverting the tube

gently. The tube was stored on ice for 20 minutes. The lysate was centrifuged at 12,000

g for 15 minutes at 4°C and the supernatant was carefully transferred to a fresh tube

avoiding any precipitate.

The plasmid DNA was precipitated by addition of 0.6 volume of isopropanol at RT for

30 'minutes. It was centrifuged at RT at 12,000 g. The pellet was washed with 70%

ethanol and dried under vacuum. It was then dissolved in 2 ml TE and collected in a

SM-24 tube. To it DNase free RNase A was added to a final concentration of 20J.Jg/ml

and incubated at 37°C for 30 minutes. The plasmid DNA was precipitated by PEG

precipitation. NaCI and PEG was added at a final concentration of 0.8M and 6.5%

respectively (A1.4.20); mixed by gentle inversion and incubated in ice-water bath for

one hour.

The solution was centrifuged at 10,000 rpm for 10 mins. The supernatant was removed

and the pellet was washed with 70% alcohol, dried and dissolved in 500!!1 of TE. This

was then transferred in a fresh micro centrifuge tube. The plasmid DNA was extracted

twice with phenol: chloroform: isoamyl alcohol, once with chloroform: isoamyl alcohol

and precipitated by adding 1/1Oth volume of sodium acetate and 2.5 volume of absolute

alcohol.

Plasmid DNA was pelleted at 12,000 g at RT for 15 minutes; washed twice with 70%

alcohol and dried under vacuum. It was dissolved in 200 J.JI TE, and estimated

spectrophotometrically. The routine yield was 400-800 J.Jg.

A2.S.2. Colony Hybridization

Screening of recombinants was also carried out using a nucleic acid hybridization

screening procedure where replica copies of the colonies were transferred on to

nitrocellulose or nylon filters (Gene Screen Plus membrane, Du Pont), lysed and after

fixing, their nucleic acid were hybridized with suitable probes. This method was

particularly used for screening recombinants where a blue/white selection was not

present. Protocol followed for plating, transferring of colonies and fixation of nucleic

acids was a modified form of what had been described previously by Sambrook et aI.,

1989.

Colonies were lifted onto nylon membrane and grown under selection condition for 2-3

hrs with colony side up. The cells were lysed by treating the filter with 1ml of 0.5N

103

Appendix 2

NaOH for 5 min and then neutralized first with 1M tris-CI pH 8.0 for 5 min and then

with 1M Tris-CI pH 8.0 and 1.5M NaCI for another 5 min.

Fixation of nucleic acids with the membrane was carried out in a UV -cross linker

(Stratagene ).

Hybridization of the membrane was then carried out following protocol described in

GeneScreen Plus manual. The blot after hybridization was exposed to X-ray film. Probe

preparation and steps involved in hybridization have been described later (A2.12).

A2.S.3. Colony PCR

This procedure was used for screening recombinants when blue white selection was not

available.

Freshly patched colonies were inoculated in 50~1 of sterile water, mixed well, boiled

for 5 min, chilled on ice for 5 mins and centrifuged at 13,000 rpm at RT for 10 mins.

2~1 of the supernatant was used as the template. Primers used for picking up the genes

were used for colony PCR and similar PCR conditions were used. A bacterial colony

carrying an empty vector was used as a negative control.

A2.6. Isolation of Genomic DNA from bacterial strains

A modification of the method of Murray and Thompson (1980) was used for DNA

extraction. Vibrio cholerae strains were inoculated in lOml LB and grown at 37°C for

16 hours. Cells were pelleted by spinning the culture at 10,000 rpm for 5 mins. The

cells were resuspended in 567~1 of TE buffer. 30~1 of 10% SDS and 3~1 of freshly

prepared proteinase K solution (20mg/ml) (A 1.4.16) were added to it. It was then

incubated at 37°C for 1 hour. 100~1 of 5M NaCI solution (A1.4.17) was added to it,

followed by the addition of 80~1 of CTABlNaCI (A1.4.15) solution heated to 65°C.

The solution was incubated at 65°C for 10mins. The DNA was extracted once with

phenol: chloroform: isoamyl alcohol (25:24:1) and once with chloroform: isoamyl

alcohol. The aqueous solution was transferred to a fresh tube and equal volume of

isopropanol was added to it. The DNA pellet was washed with 70% alcohol, dried and

resuspended in 30~1 TE.

104

Appendix 2

A2.7. Transformation of C.albicans

Transformation was done using the lithium acetate method as described by Braun and

Johnson (1997). A single well isolated colony of ura minus strain of C.albicans, was

inoculated in 10 ml of YPD supplemented with Uri dine. It was grown at 30°C at

200rpm shaking for 16-18 hrs. 1 % inoculum was added to 50ml culture and grown till

the culture reached mid log phase. The cells were then pelleted by centrifuging at 7,000

rpm at RT for 10 minutes. The pellet was resuspended in 5ml LATE buffer (A1.6.1).

The cell mix was centrifuged at 7,000 rpm for 10 minutes. The supernatant was

discarded and the cells were resuspended in 500).11 LATE buffer.

100).11 of competent cells, 5).11 of 10mg/ml sheared, single stranded, salmon sperm DNA

and 2-10 ).1g of digested DNA were mixed together. The ssDNA' was denatured by

boiling for 5 minutes and chilling on ice for 5 minutes prior to addition to the

transformation mix. The transformation mix was incubated at 30°C for 30 minutes.

Then 700).11 of PLATE buffer (A1.6.2) was added to it and vortexed for few seconds. It

was then incubated at 30°C overnight. Next day, heat shock was given at 42°C for one

hour. Cells were pelleted and washed with 1 ml of TE pH 8.0. The cells were finally

resuspended in 200 ).11 of TE. Around 100).11 of the transformation mix was plated on

appropriate plates.

A2. 7.1.Electrotransformation in Candida:

The particular strain of C.albicans to be transformed was inoculated I 5 ml YPD and

grown overnight to saturation. To 500 ml of YPD appropriate amount of inoculum was

added and grown to lxl08 cells/ml. the cells were harvested by centrifuging at 5000

rpm in SS-34 at 4°C and resuspended in 80 ml sterile water. 10 ml of 10xTE buffer

(pH7.5) was added and next 10 ml of lOx lithium acetate stock solution was sdded.

This was shaken gently at 30°C for 45 mins. 2.'5 ml IMDTT was added while swirling

and shaken gently for 15 more minutes at 30°C. The yeast suspension was diluted to

500 ml with water. The cells were washed and concentrated three times by centrifuging

at 4,000 g resuspending the successive pellets as follows:

First pellet-250 ml ice cold water

Second pellet-20-30ml ice cold sorbitol

Third pellet-O.S ml ice cold sorbitol.

105

Appendix 2

The resuspension of pellets should be vigorous each time to completely dissociate the

pellet. The final volume of resuspended cells should be 1-1.5 ml.

In a sterile, ice-cold 1.5 ml microcentrifuge tube, 40 III concentrated cells were mixed

with 100 ng of transforming DNA contained in less than 5 Ill. Pulse was given at

1.5kV,251lF and 200n parameters. 1ml ice cold sorbitol was added to recover the yeast

cells. The aliquots of yeast cells were spread directly on selection plates. Transformed

colonies appeared after 3-6 days.

A2.8. Genomic DNA Isolation from C. albicans

The C.albicans strains were inoculated in 10 ml YPD and grown at 30°C with shaking

at 200 rpm, till saturation. The cells were collected by centrifugation at 5,000 rpm for 5

minutes, resuspended in 0.5 ml distilled water and transferred to micro centrifuge tubes.

The cells were washed by giving a brief spin at 5,000 rpm for 2 minutes and the

supernatants were decanted. The pellets were resuspended in the residual liquid by

vortexing.

0.2 ml of freshly prepared breaking buffer (A1.6.4) was added to each tube. To it 0.2

ml phenol: chloroform: isoamylalcohol and 0.3 g acid-washed glass beads were added.

The cell~ were vortexed at full speed for 4 minutes and spun at 10,000 rpm for 5

minutes in a microcentrifuge. The aqueous layer was transferred to a new tube and 1ml

of chilled ethanol was added to each of them.

The tubes were spun at 10,000 rpm for 2 minutes. The supernatants were discarded ..

The pellets were wa,shed with 70% ethanol, air-dried, and resuspended in 0.4 mllX TE

(pH 8.0).

30 Ilg RNaseA (A1.4.14) was added and incubated at 37°C for 15 minutes. lOll1 4M

ammonium acetate and 1ml absolute alcohol was added to each and mixed gently by

inverting the tubes. The tubes were centrifuged at 10,000 rpm for 2 minutes. Pellets

were washed with 70% ethanol, air-dried, and resuspended in 50 III IX TE (pH 8.0).

2 III of each sample was loaded and checked on a gel, DNA quantity was estimated

visually.

A2.9. Southern Hybridization Technique

This technique was developed by Southern (1975), for detection of specific DNA

fragment among a population of digested DNA separated by gel electrophoresis.

106

Appendix 2

1. Restriction Digestion of Genomic DNA

2 Ilg of genomic DNA from C.albicans transformants were digested with appropriate

restriction enzymes in 20111 reaction volume according to the manufacturer's

instruction. 2111 of each reaction was loaded with IX EndoR and run on a 0.8% agarose

gel, to check for completion of digestion.

2. Agarose Gel Electrophoresis

The digestions, along with 1 Kb DNA ladder as marker, were loaded on a 12 cm x 14

cm 0.8% agarose gel, containing 0.5 Ilg/ml ethidium bromide. The gel was run as usual

with IX TAE buffer at 80 V constant, until the dye reached the bottom of the gel. After

electrophoresis, a tracing of the gel, and photograph, were taken to mark the positions

of the bands.

3. Southern Transfer

The gel was rinsed with MQ-water and depurination of the DNA was done by

immersing the gel in 150 ml 0.25 N Hel for 10 minutes in a baking dish, with mild

shaking on gyratory platform shaker. The solution was discarded; the gel was rinsed

with MQ-water to wash off excess Hel, and treated with 150 ml denaturing solution

(A1.7.3) for 30 minutes, under similar conditions. The solution was then exchanged

with 150 ml neutralizing solution (A1.7.4) for 30 minutes, under similar conditions.

Gene Screen Plus® membrane and 3 mm Whatman ® paper were pre-wet for few

seconds in MQ-water and equilibrated in lOX sse for 15 minutes. Gel was aligned on

top of the Whatman paper and the Gene Screen Plus® membrane, and the wells were

marked on the membrane with a needle. DNA was transferred onto the positively

charged nylon membrane by pressure blotting method, using PosiBlot Pressure Blotter

(Stratagene) at 80 mm Hg pressure for 90 minutes.

After transfer, the membrane was agitated in 0.4 N NaOH for 1 minute. This step was

performed to denature the DNA. The membrane was then neutralized in 0.2 M Tris­

Hel (pH 7.5)/IX sse for 1 minute. UV-cross linking of nucleic acids to the membrane

was done in Stratalinker.

107

Appendix 2

A2.10. RNA isolation from Candida albieans

Total RNA was isolated from 10 ml of culture either by the hot phenol method

(Ausubel et aI., 1994) or by phenol and guanidine thiocyanate method using the

monophasic TriPure Isolation Reagent (BOEHRINGER MANNHEIM).

1. Hot Phenol Method

The cell pellets from various conditions were collected in microfuge tubes. The pellets

were resuspended in TES buffer (A1.S.1) to which 250 III phenol saturated in DEPC

treated water was added and vortexed. The mixture was heated at 65°C for 1 hour with

intermittent vortexing. The hot mixture was then chilled on ice for 5 minutes. 200 III

TES was added to the mixture and vortexed vigorously again. The mixture was then

centrifuged at 12000 g for 10 minutes at RT. The proteins and unlysed cell debris

formed a thick interphase, above the organic phase. The aqueous phase was carefully

removed to another tube, without disturbing the interphase, even if it meant leaving

some of the aqueous phase behind. The aqueous phase was extracted with chloroform

once, and the RNA was precipitated by adding 1/6th volume 2M NaOAc pH 4.2

(A1.S.3) and twice the volume ethanol. The RNA was recovered by centrifugation at

12000 g for 10 minutes at RT in a microfuge. The pellet was washed in 70% ethanol in

DEPC water, dried and dissolved in DEPC treated water.

2. TriPure Method of RNA Isolation

TriPure isolation reagent was added to the polypropylene tubes containing cell pellet at

room temperature. 1 ml reagent was used for each ~107 cells. Equal volumes of 0.45

mm chilled glass beads were added and homogenized for 3 minutes on cyclomixer.

This was incubated at room temperature for 5 minutes to ensure complete dissociation

of the nucleoprotein comp\exes. For phase separation, chloroform was added, 0.2 ml

for each 1 ml of TriPure reagent used. The tubes were capped, vortexed vigorously for

15 seconds and incubated at room temperature for 10 minutes. The tubes were

centrifuged at 12,000 g for 15 minutes at 4°C, to separate the solution into three phases.

The colourless upper aqueous phase was collected in a new tube and the RNA was

precipitated by adding isopropanol (0.5 ml for each 1 ml TriPure reagent used) and

incubated at room temperature for 10 minutes. RNA was pelleted down by centrifuging

the tubes at 12,000 g for 10 minutes at 4°C; supernatant was discarded and the pellet

lOS

Appendix 2

was given 75% ethanol wash. It was then air-dried and dissolved in DEPC-treated

RNase free water incubating at 55 DC for 10 minutes.

A2.11. Agarose gel electrophoresis of RNA and Northern blotting

Formaldehyde-denatured RNA gel was prepared as described by Sambrook et al.

(1989), formaldehyde (Glaxo) pH 3-3.5, was used. Formamide was deionised and

stored in small aliquots at _20DC.

RNA samples (30lJg) 10 1J1

10xMOPS 3 1J1

Formamide 10 1J1

Formaldehyde 6 Ml

The above components were mixed and incubated at 60 DC for 15 minutes and then

chilled on ice. 2 1J110ading buffer (A1.8.4) was added to the above mix.

1.5% (WN) agarose gel containing IX MOPS and 2.2M formaldehyde was prepared.

The gel was placed in electrophoresis tank with IX MOPS buffer (A1.8.5) for 15

minutes prior to loading. The denatured RNA was loaded in the wells and was left for

15 minutes to enable the formaldehyde to diffuse in the gel. The gel was run at 80V till

the bromophenol blue ran 2/3 distance (3-4 hours).

After electrophoresis, the gel was rinsed thrice in five times the gel volume of DEPC

treated water, followed by RNA hydrolysis by soaking in 50mM NaOH, lOmM NaCI

for 45 minutes. The gel was then neutralized in O.IM Tris-CI pH 7.5. The RNA was

transferred to gene screen plus membrane using 10XSSC as described (gene screen

Plus protocols, NEN), and baked for 2 hours at 80D C to reverse formaldehyde reaction. \

The amount of RNA loaded was verified by ethidium bromide staining (Sambrook et.

aI., 1989) of parallel lanes.

A2.12. Hybridization

A2.12.1. Prehybridization

The membrane was pre-wet in 2X SSC for 1 minute and then placed in a hybridization

bottle and prehybridized overnight, with 50 Mlprehybridization buffer (A1.9.1) per cm2

membrane (~9 ml for 12 cm x 14 cm membrane), at 42DC.

109

A2.12.2. Preparation of Probe by Random Priming Reaction

1. Labeling reaction

Labeling was done according to Feinberg and Vogel stein (1983).

Appendix 2

25 ng template DNA fragment was taken in a volume of 17.5IJ.I sterile MQ-water. It

was denatured in boiling water bath for 5 minutes and quickly placed in ice for 5

minutes and centrifuged briefly in the cold. The following reagents were added to the

DNA in the order listed:

2.5IJ.llOX Labeling Buffer which includes Random Octadeoxyribonucleotides

3IJ.I dNTP mixture (lIJ.I ofdATP, dTTP, and dGTP)

1IJ.I a 32p dCTP (3,000 ci/mmol, 50 IJ.Ci)

1IJ.I DNA Polymerase I-Klenow fragment (5 units)

Total reaction volume was 25 IJ.l.

The reaction mix was incubated at 37°C for 1 hour or 30°C overnight. The reaction was

terminated by adding 5 IJ.I of 0.2 M EDTA and stored at -20°C.

2. Determination of % Incorporation

1IJ.I of the reaction sample was diluted to 100IJ.I with O.2M EDTA. 5IJ.I of the diluted

sample was spotted onto each of two DE-81 filters. One of the filters was used to

measure total radioactivity in the reaction and was not subsequently washed. The other

filter was used to measure radioactivity incorporated. The filters were dried under a

heat lamp for 10 minutes. One filter was added to a beaker containing 100ml of 0.5M

Na2HP04 and washed for 5 minutes, with mild agitation. Washing was repeated twice

using fresh 0.5M Na2HP04. The filter was then washed twice with MQ-water and twice

with ethanol. The filter was dried under a heat lamp for I? minutes and placed in a

scintillation vial. The cerenkov counts of the filters were read and the ratio of washed to

unwashed counts was calculated which gave the % incorporation.

3. Purification of Labeled Probes

Probes synthesized were separated from unincorporated nucleotides by filtration

through Sephadex® G-50 spin column. The reaction volume was increased to 200 IJ.I

with IX TE (pH 8.0) before purification.

110

Appendix 2

A2.12.3. Preparation of Probe Solution for Hybridization

For 9 ml prehybridization buffer, 900 !J,l probe solution was prepared. To the 200 !J,l

probe solution, salmon sperm DNA was added at a concentration of 0.5 mg/ml, and the

volume was made upto 900 !J,l with sterile MQ-water. The probe solution was ,

denatured by heating for 10 minutes in a boiling water bath and chilled on ice for 15

minutes before adding to the prehybridization buffer.

A2.12.4. Hybridization

Hybridization was carried out in hybridization bottles, at 42°C, for 16 hours III a

hybridization oven.

A2.12.S. Post-hybridization Washing

The membrane was washed with 2X SSC at room temperature for 5 minutes. The

second washing was with 2X SSC/l % SDS at 42°C for 20 minutes. The washing was

repeated depending on the count. The membrane was then washed with 0.2X SSCII %

SDS at 42°C for 20 minutes. The washing was repeated if required. After each wash the

background count was monitored with a Hand Monitor to avoid washing off

specifically bound signal.

A2.12.6. Exposing and Developing of Film

The membrane was wrapped securely in a saran wrap, and marked with fluorescent ink

at different positions to align the blot on the film later. The blot was exposed to Kodak

X-Omat™ film in a film cassette, and incubated at -80°C for 2 hours to 2 days

depending on the count. The film was developed, aligned with the blot and tracing, to

mark the positions of the bands.

Alternatively, the blots can be exposed in phospho imager.

A2.13.Western Blotting

The cells containing· the myc-epitopes were lysed. The lysates were prepared for gel

electrophoresis. SDS gel electropohoresis was done with the lysate. The protein was

transferred to a PVDF membrane in transfer buffer containing 20% methanol, 24 mM

Tris base, 194 mM glycine. The membrane was transferred to a tray and incubated for 1

hour at +15 to +25°C (or overnight at 4°C) with a 1:10 dilution OfW@gt~1'\'\ RlMkins

111

Appendix 2

Reagent (Cat. No. II 921 673 00 I) in phosphate buffered saline with 0.1 % Tween 20,

pH 7.5 (PBST). The membrane was washed three times with PBST. The Anti-c-myc

antibody concentrate was diluted to I lJg/ml in a I :20 dilution of Western Blocking

Reagent. The membrane was incubated with this diluted Anti-cmyc antibody for 1-2

hours at + 15 to +25°C with gentle rotation. The membrane was washed three times

with Ix WashBuffer .Goat Anti-Mouse IgG (H+L) HRP Conjugate was diluted 1 :4000

into a 1 :20 dilution of Western Blocking Reagent. The membrane was incubated with

this diluted antibody solution for 1 hour at R T with gentle rotation. The membrane was

again washed three times with Ix Wash Buffer. Detection solutions A and B

(Amersham ECL plus Western Blotting Detection Reagent) were mixed in a ratio of

40: 1. The excess wash buffer was drained from the membranes and the membrane was

placed on a clean Saran wrap with the protein side up. The mixed detection reagent was

pipetted onto the membrane and incubated for 5 mins at room temperature. The

membrane was exposed to X-ray film and developed within 1-5 mins interval.

A.2.14.1. Preparation of Sphaeroplasts:

Cell pellet was resuspended in 2-3 volumes Ice -cold water and immediately

centrifuged at 3500 rpm, 4°C. Supernatant was discarded and the cell pellet was

resuspended by adding 1 vol. of Zymolase buffer containing 30mM DTT and incubated

for 15 min at room temperature.( This step facilitated the subsequent zymolase

treatement and sphaeroplast lysis by breaking disulphite bonds). It was again

centrifuged for 5 min at 1500g and resuspended in 3 vol of zymolase buffer. 200 U of

lyticase was added for 1 vol. of original cell pellet to the resuspended cells. Incubation

was done for 2 hours at 30°C on a shaker plat form at ~ 50 rpm. The complete

conversion of cells to sphaeroplast was confirmed by checking under microscope. The

sphaeroplasts were centrifuged for 5 min at 1500g at 4°C. The supernatant was

discarded carefully. The sphaeroplast pellet was washed gently by resuspending the

pellet in 2 volumes of ice cold zymolase buffer and centrifuged for 5 min at 3500 rpm

at 4°C. This step w~~ r~p€:ated fQ[ 2 mom [jmE~: {I!~: ~~hifi[ ~t{;n $iili h;u9-~~~V-..\ ,~.

f8rnlJ<!flfl«' F~;g.(:gl',i:;;,i, ~1~g~B1itli~~D;i nm:l m.l!i;J~tlnf1o~a}, t:::J I .... w .... b~r.Ja l:i" '-.. _. "",.,." ..... .., . 0" •

112

Appendix 2

A.2.14.2. Nuclei Preparation by Differential Centrifugation:

Nuclei suitable for chromatin studies and I or nuclear protein extracts were prepared by

osmotically lysing sphaeroplasts in presence of Ficoll, which preserves nuclear

structure and prevents proteins from leaking out of the nucleus, followed by

differentnial centrifugation. Cells were resuspended in 0.5 vol. of zymolase buffer. The

resuspended cells were added drop by drop into beaker containing 20 vol. of ice cold

Ficoll buffer with continuous stirring in an ice-bath. After adding completely, the

stirring process was continued for 2 more hours. The suspension was transferred to

centrifuge tubes and centrifuged for 5 min at 5000 rpm and 4 DC to pellet cell debris

and unlysed sphaeroplasts. This step was repeated for 4 times till no pellet was formed,

which indicated that nuclei suspension became free of debris and unlysed

sphaeroplasts. The supernatant was transferred to fresh centrifuge tubes and centrifuged

for 20 min at 13,000 rpm and 4DC. The supernatant was discarded; the pellet with halo

that was formed contained the nuclei.

113