APPENDIX-I
1. Media used
1.1. Murashige and Skoog medium
Components
*NH4NO3
1.2. Modified B5 medium
Concentration
(mg/l)
Components
Concentration
(mg/l)
1650
*KNO3
2500
134
1900
M1
M1
440
M2
150
M3
370
M3
250
M4
KH2PO4
170
M4
150
m2
H3BO3
6.2
m1
H3BO3
3
m3
MnSO4.4H2O
22.3
m2
CoCl2.6H2O
0.025
m4
ZnSO4.7H2O
8.6
m3
CuSO4.5H2O
0.025
m1
KI
0.83
m4
Na2MoO4.2H2O
0.25
*KNO3
*CaCl2.2H2O
MgSO4.7H2O
(NH4)2SO4
CaCl2.2H2O
MgSO4.7H2O
NaH2PO4.H2O
m5
Na2MoO4.2H2O
0.25
m5
m6
CuSO4.5H2O
0.025
CoCl2.6H2O
FeSO4.7H2O
ZnSO4.7H2O
2
m6
MnSO4.H2O
10
0.025
m7
KI
0.75
M2
27.8
V1
Nicotinic acid
1
M1
Na2EDTA.2H2O
37.3
V2
1
V2
Meso-inositol
100
V3
10
V4
0.5
V4
Meso-inositol
100
V5
0.1
Na2EDTA.2H2O
37.3
V3
0.5
FeSO4.7H2O
27.8
V1
Glycine
2.0
Sucrose
20 g/l
Sucrose
30 g/l
m7
Pyridoxine-HCl
Thiamine-HCl
Nicotinic acid
Pyridoxine-HCl
Thiamine-HCl
Appendices
Depending upon the solubility of the salts, separate stocks are made for Ammonium
nitrate (*), Potassium nitrate (*), Vitamins (v1-v5), Major elements (M1-M5) and Minor
elements (m1-m6).
Solubility of ionic compounds in water
Negative Ions
(Anions)
+
Positive Ions
(Cations)
=
Solubility of
compounds
in water
Any anion
+
alkali ions
(Li+, Na+, K+, Rb+, Cs+,
Fr+)
=
soluble
sodium fluoride (NaF)
is soluble
Any anion
+
hydrogen ion
[H+(aq)]
=
soluble
hydrogen
chloride
(HCl) is soluble
Any anion
+
=
soluble
Nitrate
NO3-
+
ammonium ion
(NH4+)
any cation
=
soluble
ammonium
chloride
(NH4Cl) is soluble
potassium
nitrate
(KNO3) is soluble
Acetate
(CH3COO-)
+
any cation
=
soluble
Chloride (Cl-),
Bromide (Br-),
Iodide (I-)
+
silver
(Ag+),
lead
+
(Pb ), mercury (Hg2+),
Copper (Cu+), thallium
(Tl+)
any other cation
=
low solubility
(insoluble)
=
soluble
calcium
(Ca2+),
strontium
(Sr2+),
2+
barium (Ba ), silver
(Ag+), lead (Pb2+),
radium (Ra2+)
any other cation
=
low solubility
(insoluble)
barium
sulphate
(BaSO4) forms a white
precipitate
(a white
solid)
=
soluble
copper
sulphate
(CuSO4) is soluble
alkali ions (Li+, Na+,
K+, Rb+, Cs+, Fr+),
alkali earth metals
(Be2+, Mg2+, Ca2+, Sr2+,
Ba2+,
Ra2+),
and
+
H (aq), NH4+
any other cation
=
soluble
magnesium sulphide
(MgS) is soluble
=
low solubility
(insoluble)
zinc sulphide (ZnS) is
insoluble
hydroxide OH- + alkali
ions (Li+, Na+, K+, Rb+,
Cs+, Fr+), H+ (aq),
NH4+, Sr2+, Ba2+, Ra2+,
Tl+
any other cation
=
soluble
strontium
hydroxide
{Sr(OH)2} is soluble
=
low solubility
(insoluble)
alkali ions (Li+, Na+,
K+, Rb+, Cs+, Fr+), H+
(aq), NH4+
any other cation
=
soluble
silver
hydroxide
(AgOH) is insoluble
(forms a precipitate)
ammonium phosphate
{(NH4)3PO4} is soluble
=
low solubility
(insoluble)
+
Sulphate (SO42-)
+
+
Sulfide (S2-)
+
+
Hydroxide (OH-)
+
+
Phosphate,
PO43-,
Carbonate,
CO32-,
2Sulphite, SO3
+
+
ii
Example
sodium
acetate
(CH3COONa)
is
soluble
silver chloride (AgCl)
forms
a
white
precipitate (a white
solid)
potassium
bromide
(KBr) is soluble
magnesium carbonate
(MgCO3) is insoluble
Appendices

All compounds of the ammonium ion (NH4+) and of alkali metal (Group IA)
cations are soluble.

All nitrates and acetates (ethanoates) are soluble.

All chlorides, bromides and iodides are soluble EXCEPT those of silver, lead and
mercury (I).

All sulphates are soluble EXCEPT those of silver, lead, mercury (I), barium,
strontium and calcium.

All carbonates, sulfites and phosphates are insoluble EXCEPT those of
ammonium and alkali metal (Group IA) cations.

All hydroxides are insoluble EXCEPT those of ammonium, barium and alkali
metal (Group I) cations.

All sulfides are insoluble EXCEPT those of ammonium, alkali metal (Group I)
cations and alkali earth metal (Group II) cations.

All oxides are insoluble EXCEPT those of calcium, barium and alkali metal
(Group I) cations; these soluble ones actually react with the water to form
hydroxides (hydrolyse).
1.3.
Yeast extract mannitol broth
For 1L of liquid YEMB (pH 7)
YEMB medium (pH 7)
100 ml of Stock A
Dissolve 5 g K2HPO4 and 1 g of NaCl Stock A: 10 ml
in 100 ml distilled water
Stock B: 0.8 ml
Yeast extract: 1 g
100 ml of Stock B
For
1mM
MgSO4.7H2O
Mannitol: 10 g
dissolve
For solid add 15 g/l of Bacto-agar
0.0246 g MgSO4.7H2O in 100 ml
distilled water
1.4.
Luria bertani
 Bacto tryptone - 10 g/l
 Bacto yeast extract - 5 g/l
 NaCl - 10 g/l
Adjust pH to 7; add 15 g/l Bacto-agar for solid medium
iii
Appendices
2. Reagents and components
2.1. Hoagland solution
 KNO3 - 506 mg/l
 CaNO3.4H2O - 1180 mg/l
 MgSO4.7H2O - 493 mg/l
 KH2PO4 - 136 mg/l
2.2. GUS assay buffer (for 10 ml)
 Sodium phosphate buffer (pH 7; 100 mM) - 2 ml (from 500 mM stock)
 Potassium ferrocyanide (1 mM) - 100 µl (from 100 mM stock;
Mw 368.35; 3.68 g/100 ml)
 Potassium ferricyanide (1 mM) - 100 µl (from 100 mM stock;
Mw 329.24; 3.29 g/100 ml)
 EDTA (pH 8; 10 mM) - 200 µl (from 0.5 M stock; Mw 372.2;
18.61 g/100 ml)
 Triton-X 100 (0.1% v/v) - 10 µl
 5-Bromo, 4-chloro, 3-indolyl, -D-glucuronide cyclohexylammonium
salt, (Sigma Aldrich, USA) (1 mg/ml) - 10 mg
 Chloramphenicol (100 µg/ml) - 100 µl (from 100 mg/10 ml stock)
ADW to make the final volume to 10 ml. Store at 4°C
2.3. Colony lysate buffer (for 10 ml)
 Tris-HCl (pH 8; 10 mM) - 0.1 ml (from 1 M stock)
 EDTA (pH 8; 1 mM) - 0.02 ml (from 0.5 M stock)
 Tween-20 (0.1% v/v) - 0.01 ml
Buffer sterilized by autoclaving. Single bacterial colony is added
to 50 µl of buffer and boiled for 10 min, centrifuged and
supernatant is used as colony lysate for PCR.
iv
Appendices
2.4. Galbraith’s nuclei isolation buffer (pH 7, for 100 ml)
 Magnesium chloride (45 mM) - 0.4284 g (Mw 95.211)
 Sodium citrate (30 mM) - 0.7742 g (Mw 258.069)
 3-(N-Morpholino) propane sulfonic acid (MOPS; 20 mM) - 0.4185 g
(Mw 209.2633)
 Triton-X 100 (0.1% w/v) - 0.1 g
2.5. Acid ninhydrin
Ninhydrin (Sigma, USA; 1.25 g) dissolved in a mixture of 30 ml glacial acetic
acid and 20 ml of 6 M ortho phosphoric acid (7.636 ml ortho phosphoric acid in
12.36 ml of milliQ water) by warming at 40°C. Kept at 4°C and used within 24 h.
2.6. SOD homogenization buffer (10 ml)
 EDTA (pH 8; 2 mM) - 40 µl (from 0.5 M stock; 18.6 g/100 ml of 50 mM
potassium phosphate buffer pH 7.8)
 DTT (1 mM) - 1 ml (from 10 mM stock; 0.015 g/10 ml of 50 mM
potassium phosphate buffer pH 7.8)
 PMSF (1 mM) - 1ml (from 10 mM stock; 0.017 g/10 ml of 50 mM
potassium phosphate buffer pH 7.8; first dissolved in iso-propanol and
then volume made up in buffer; always prepared fresh)
 Triton X-100 (0.5% v/v) - 1.66 ml (from 3% v/v stock; 3 ml per 100 ml
of 50 mM potassium phosphate buffer pH 7.8)
 PVPP (5% w/v) - added 50 mg while grinding
 Potassium phosphate buffer (pH 7.8; 50 mM) - 6.3 ml to make the final
volume to 10 ml
2.7. SOD enzyme assay stocks

Methionine - 0.7385 g/25 ml of 50 mM of potassium phosphate buffer
(pH 7.8)

Triton X-100 - 0.25 ml/100 ml of 50 mM of potassium phosphate buffer
(pH 7.8)

NBT - 3.498 mg/2.5 ml of 50 mM of potassium phosphate buffer
(pH 7.8); prepared fresh
v
Appendices

Riboflavin - 3.3 mg/100 ml of 50 mM potassium phosphate buffer (pH 7.8);
prepared fresh
for 1 run
(µl)
2.67
5
6.67
10
10
Rxn mixture
Riboflavin
Triton X-100
NBT
Methionine
Sample
50 mM potassium phosphate buffer (pH 7.8) to make the final volume to 200 µl
2.8. APX homogenization buffer

EDTA (2 mM; pH 8) - 40 µl (from 0.5 M stock; 18.6 g/100 ml of 50 mM
sodium phosphate buffer pH 7)

DTT (1 mM) - 1 ml (from 10 mM stock; 0.015 g/10 ml of 50 mM sodium
phosphate buffer pH 7)

PMSF (1 mM) - 1 ml (from 10 mM stock; 0.017 g/10 ml of 50 mM sodium
phosphate buffer pH 7; first dissolved in iso-propanol and then volume made
up in buffer, always prepared fresh)

Ascorbate (3 mM) - 3 ml (from 10 mM stock; 17.5 mg/10 ml of 50 mM
sodium phosphate buffer pH 7)

Triton X-100 (0.5% v/v) - 1.66 ml (from 3% v/v stock; 3 ml/100 ml of
50 mM sodium phosphate buffer pH 7)

PVPP (5% w/v) - added 50 mg while grinding

Sodium phosphate buffer (pH 7; 50 mM) - 3.3 ml to make the final volume
to 10 ml
2.9. APX enzyme assay stocks

EDTA (0.1 mM) - 0.5 ml (from 10 mM stock; prepared in 50 mM sodium
phosphate buffer pH 7)

Ascorbate (0.5 mM) - 1 ml (from 5 mM stock; 4.4 mg/5 ml of sodium
phosphate buffer pH 7)
vi
Appendices

H2O2 (1 mM) - 1 ml (from 10 mM stock; 10.2 µl from bottle which is 9.8 M
+ 9.89 ml milliQ water), added just before read in micro plate reader and vial
covered with Al foil
Total volume made to 10 ml by adding 7.5 ml of 50 mM sodium
phosphate buffer pH 7. For assay, 20 µl of sample (enzyme) is
added to 180 µl of above rxn mix.
2.10. IEF solutions
2.10.1. Sample buffer (pH 6.8)
 Tris-HCl (0.0625 M; 0.7571 g)
 SDS (2% w/v; 2 g)
 Sucrose (10% w/v; 10 g)
Tris (0.7571 g) dissolved in approx 40 ml of milliQ H2O and pH
maintained at 6.8 with HCl. To the above mix, added 10 g
sucrose,
2 g SDS and kept in the hot air oven (at 30-40°C) to
allow SDS to dissolve. Volume made to 100 ml with milliQ
water. To 47.5 ml of the above solution added 2.5 ml
β-mercaptoethanol and 1 mg bromophenol blue (0.002% w/v).
2.10.2. Bradford reagent
Added 50 mg of Commassie Brilliant Blue G250 (Sigma, USA) in 50 ml of
methanol and 100 ml of 85% H3PO4 (Phosphoric acid) to the solution and
made up the volume up to 500 ml with milliQ H2O. Filter to remove
precipitates. Added an additional 350 ml of distilled water and stored at 4°C.
The solution is light sensitive and was therefore, prepared and stored in dark.
The solution should be used within one month as it degrades.
2.10.3. Urea/ thiourea lysis solution (pH 7.4) in DW (for 100 ml)
 Urea * (7 M) - 42.042 g (Mw 60.06)
 Thiourea (2 M) - 15.224 g (Mw 76.12)
 CHAPS (4% w/v) - 4 g
 Tris-HCl (18 mM) - 0.2837 g (Mw 157.6)
vii
Appendices
 Trizma-base (14 mM) - 0.1696 g (Mw 121.14)
 Triton-X 100 (0.2% v/v) - 0.2 ml
 Pharmalyte (pH 3-10) (1% v/v) (if available)
 Protease inhibitor (if necessary; 50 U/ml)
* If necessary, the concentration of urea can be increased to 9 or 9.8 M
† Other detergents (Triton X-100, NP-40, and other non-ionic or zwitterionic detergents)
can be used instead of CHAPS
Note: Protease inhibitors may be added if necessary
2.10.4. Rehydration buffer (store in aliquots at -20°C; for 100 ml)

Urea (8 M) - 48.05 g (Mw 60.06)

3-[(3-Cholamidopropyl)dimethylammonio]-1-propane
sulfonate
(CHAPS 2% w/v) - 2 g

Bromophenol blue (0.002% w/v) - 2 mg

DTT* (20 mM, added prior to use) - 0.709 g (Mw 354.49)

IPG buffer (0.5% v/v) (added prior to use) - 0.5 ml
2.10.5. Bromophenol blue stock solution

Bromophenol blue (1% w/v) - 100 mg

Tris-base 50 mM - 60 mg
Double distilled H2O to make the final volume to 10 ml
* DTT is added just prior to use: 2.8 mg DTT per 1 ml aliquot of rehydration stock
solution. For rehydration loading, sample is also added to the 1ml aliquot of rehydration
solution just prior to use
†† Use Pharmalyte 3-10 for Immobiline DryStrip 3-10 or 3-10 NL, Pharmalyte 5-8 for
Immobiline DryStrip 4-7
2.10.6. SDS equilibration buffer (store at -20°C)
Final concentration amount

Tris-HCl (pH 8.8; 50 mM) - 5 ml (from 1 M stock; Mw 157.6)

Urea (6 M) - 36.03 g (Mw 60.06)

Glycerol (30% v/v) - 34.48 ml (from 87% v/v bottle stock)

SDS (2% w/v) - 2 g (Mw 288.38)

Bromophenol blue (0.002% w/v) - 400 µl of 1% solution
viii
Appendices
Double distilled H2O to make the final volume to 100 ml

DTT (50 mM) - 1.772 g (Mw 354.49; added prior to use)

Iodoacetamide (4% w/v) - 4 g (added prior to use)
2.11. SDS-PAGE
The acrylamide, N, N'-methylenebisacrylamide, TEMED, ammonium persulfate
and SDS in this appendix are extremely hazardous. You should have a
manufacturer's safety data sheet (MSDS) detailing the properties and precautions
for all chemicals in your lab. The safety sheets should be reviewed prior to
starting the procedures in this manual. General handling procedures for
hazardous chemicals include using double latex gloves for all protocols.
Hazardous materials should be weighed in a fume hood while wearing a
disposable dust mask.
2.11.1. 30% T, 2.6% C Monomer stock solution
(30% acrylamide, 0.8% N, N'-methylenebisacrylamide; 200 ml)
Final concentration amount

Acrylamide (30% w/v) - 60 g (Mw 71.08)

N, N'-Methylenebisacrylamide (0.8% w/v) - 1.6 g (Mw 154.17)
Double distilled H2O to make the final volume to 200 ml. Store
at 4°C in dark
2.11.2. Resolving gel buffer
(3 M Tris-HCl pH 8; 100 ml)
Final concentration amount

Tris-base (3 M) - 36.3 g (Mw 121.1)
Double distilled H2O - 40 ml

HCl (Mw 36.46) to adjust pH 8
Double distilled H2O to make the final volume to 100 ml. Store
at 4°C
2.11.3. Stacking gel buffer (SGB; pH 6.8)
(0.5 M Tris-HCl pH 6.8; 100 ml)
Final concentration amount
ix
Appendices
 Tris-base 0.5 M - 6 g (Mw 121.1)
Double distilled H2O - 400 ml
 HCl (Mw 36.46) to adjust pH 6.8
Double distilled H2O to make the final volume to 100 ml. Store
at 4°C
2.11.4. 10% SDS
Final concentration amount
 SDS (10% w/v) - 5 g (Mw 288.38)
Double distilled H2O to make the final volume to 50 ml. Store
at room temperature
2.11.5. 1.5% Ammonium persulfate
Final concentration amount

Ammonium persulfate (1.5% w/v) - 0.15 g (Mw 228.20)
Double distilled H2O to make the final volume to 10 ml and store at
-20°C
Fresh ammonium persulfate "crackles" when water is added. If it does not, replace it
with fresh stock. Prepare just prior to use
2.11.6. TEMED
150 µl in 10 ml double distilled H2O and store at -20°C
2.11.7. Gel storage solution
7% Glacial acetic acid
2.11.8. SDS electrophoresis buffer*
(25 mM Tris-HCl pH 8.3; 192 mM Glycine; 0.1% SDS; For 10 l)
Final concentration amount
 Tris-base (25 mM) - 30.3 g (Mw 121.1)
 Glycine (192 mM) - 144 g (Mw 75.07)
 SDS (0.1% w/v) - 10 g (Mw 288.38)
Double distilled H2O to make the final volume to 10 l
x
Appendices
*Because the pH of this solution need not be checked, it can be made up directly in large
reagent bottles. Store at room temperature
2.11.9. Agarose sealing solution
Final concentration amount
 SDS electrophoresis buffer - 100 ml
 Agarose (0.5% w/v) - 0.5 g
 Bromophenol blue (0.002% v/v) - 200 µl
Add all the ingredients into a 500 ml Erlenmeyer flask. Swirl to
disperse. Heat in a microwave oven on low or a heating stirrer
until the agarose is completely dissolved. Do not allow the
solution to boil over. Dispense 2 ml aliquots into screw-cap tubes
and store at room temperature.
2.11.10. 12% Resolving gel (7.5 ml)
 Acrylamide - 3 ml
 Resolving gel buffer - 0.95 ml
 Ammonium persulfate - 0.375 ml
 SDS (10%) - 0.075 ml
 TEMED - 0.25 ml
Double distilled H2O to make the final volume to 7.5 ml
2.11.11. 4.5% Stacking gel
 Acrylamide - 0.75 ml
 Stacking gel buffer - 1.25 ml
 Ammonium persulfate - 0.25 ml
 SDS (10%) - 0.05 ml
 TEMED - 0.25 ml
Double distilled H2O to make the final volume to 5 ml
2.11.12. Staining solution
 Methanol - 100 ml
 Glacial acetic acid - 20 ml
xi
Appendices

CBB R-250 (0.1% w/v) - 220 mg

Double distilled H2O (100 ml)
Mix, filter and used for staining the gel overnight.
2.11.13. Destaining solution
 Same as staining solution but without CBB R-250
2.12. Propidium iodide (PI) stock
5 mg PI in 10 ml double distilled H2O
Vortexed and stored at 4°C
2.13. IPTG
Make a solution of IPTG (Isopropylthio-β-D-galactoside; Mw 238.3) by
dissolving 2 g of IPTG in 8 ml of water. Adjust the volume of the solution to
10 ml with water and filter sterilized. Dispense the solution into 1 ml aliquots
and store them at -20°C (used 4 µl).
2.14. X-gal
Dissolve
X-gal
(5-Bromo-4-chloro-3-indolyl--D-galactopyranoside)
in
dimethylformamide to make a 20 mg/ml solution. Use a glass or polypropylene
tube. Wrap the tube containing the solution in aluminium foil to prevent damage
by light and store at -20°C. It is not necessary to sterilize X-gal solution by
filtration (used 40µl).
xii