Appendix - I
A)
Buffers and Reagents
1.
Borate buffer, 0.1M pH 7.4:
Boric acid
- 24.732 gm
in 4 lit of D.D.W
Borax
- 19.07 gm
in 500 ml of D.D.W
Add borax solution to boric acid solution slowly to achieve
pH 7.4.
2.
Carbonate-bicarbonate buffer 0.06M,
pH 9.6
Anhydrous sodium carbonate (Na2CO3) -
1.15 g
Sodium bicarbonate (NaHCO3)
- 4.13 g
Sodium azide
- 0.2 g
Dissolve in 1000 ml distilled water and adjust pH to 9.6
3.
Carbonate - bicarbonate buffer 1M,
pH 9.5
Anhydrous sodium carbonate (Na2CO3)
- 31 g
Sodium bicarbonate (NaHCO3)
- 13.78 g
Sodium azide
- 0.2 g
Dissolve in 1000 ml distilled water after adjusting the pH to
9.5
4.
Carbonate bicarbonate buffer- 0.1M,
pH 10.6
Anhydrous Sodium carbonate (Na2CO3)
- 1.80 g
Sodium bicarbonate (NaHCO3)
- 252 mg
Dissolve in 400ml double distilled water and adjust the pH.
5.
Citrate Phosphate buffer (pH 5.0)
Citric acid
- 9.6 g in
500 ml distilled water
Sodium phosphate (dibasic)
- 14.2 g in
500 ml distilled water
Add phosphate solution to the citric acid solution until the
pH is 5.0 dilute 1:1 with double distilled water
6.
Citrate phosphate buffer: 0.1M, pH
5.6
Citric acid
- 21.01g in 1lit
double distilled water.
Sodium phosphate (dibasic)
- 14.2 g in 1lit
double distilled water.
Adjust the pH of Sodium Phosphate solution to pH 5.6 by
adding citric acid solution.
7.
2
M Ethanolamine - Take
1.25 ml of ethanolamine
& make upto 10 ml with doubled
ouble distilled water.
8.
Ethidium bromide : Add 1g of
Ethidium bromide to 100 ml of Distilled water. Stirr on a
magnetic stirrer for several hours to ensure that the dye has
dissolved completely. Wrap the
container in aluminium foil or transfer to a dark bottle & store at 40
C.
9.
0.1 M Glycine HCl buffer, pH 2.8
Glycine
- 1.50 g
HCl
- 0.2 M
(32.4 ml)
Dissolve in 50 ml double distilled water, add 32.4 ml of 0.2
M HCl and adjust the volume
to
200 ml.
10. Gel loading buffer: (6X buffer)
0.25 % bromophenol blue
0.25 % Xylene cynol
30 % Glycerol in Water
11. Oligonucleotide Primers : ( For
Excretory - Secretory Antigen gene
)
Forward Primer: 5¢ AAT CTA ACC GGT GAC GGA AAAG 3¢
Reverse Primer:
5¢ TTG GGC ATC CGT TAA GCC AAC
TTA 3¢
12.Phosphate Buffer Saline 0.01 M, pH 7.2
0.25
M SPB
- 40 ml
NaCl
- 9g
Distilled
Water
- 960 ml
Mix
and adjust the pH.
13. Phosphate
buffer saline 0.05 M pH 7.2
Make
up 100 ml of 0.25 M SPB to 500 ml with double distilled water and add 4.5 g of
NaCl.
14. Phosphate
buffer saline 0.05 M pH 8.00
Na2Hpo4
- 237.36 g
Nah2po4
- 104.00 g
Nacl
- 9 g
Dissolve
in 500 ml of D.D.W. adjust pH to
8.00 and make up to 1000ml with D.D.W.
15. PBS
with 0.05% Tween -20 (PBS/T)
Add
0.5 ml of Tween - 20 to one litre of PBS (0.01 M).
16. Sodium
acetate buffer 0.1 M, pH 4.5 with NaCl (0.1 M)
Acetic
Acid
- 1.75 ml
Sodium
acetate
- 1.6 g
NaCl
- 10 g
Dissolve
in 100 ml double distilled water and make upto 500 ml.
17. Sodium
acetate buffer 1.0 mM pH 4.4:
Solution
A
- 8.24 gm
sodium acetate anhydrous
in1 lit D.D.W.
Solution
B
- 6.005 ml
glacial acetic acid in 1 lit.
D.D.W.
Add
one part of solution A & two part of solution B & dilute 1:100 with
D.D.W
18. Sodium
carbonate bicarbonate buffer 1 pH 9.5
Na2CO3
- 1.59 gm
NaHCO3
- 2.93 gm
Dissolve
in 1 lit of D.D.W & adjust pH
19. Sodium
carbonate bicarbonate buffer 2 pH 9.5 (0.2M)
Na2CO3
-
21.2 gm in 1 lit of D.D.W-solution A
NaHCO3
-
16.8 gm in 1 lit of D.D.W-solution B
Add
slowly solution A to solution B to achieve pH 9.5
20. Sodium
Phosphate Buffer (SPB). pH 7.2 (0.25M).
Sodium
Phosphate dibasic (Na2HPO4)
- 29.67 g
Sodium
dihydrogen Orthophosphate
(NaH2PO4)
- 13.00 g
Dissolve
in 1000 ml double distilled water and adjust pH.
21. SPB
pH 6.8 (0.1 M)
Make up 400 ml of 0.25 M SPB to 1000 ml with double distilled
water after adjusting
the pH to 6.8
22. SPB
pH 7.2 (0.05 M)
Make
up 200 ml of 0.25 M SPB to 1000 ml with double distilled water.
23. SPB
pH 7.2 (0.01 M)
Make
up 40 ml of 0.25 M SPB to 1000 ml with double distilled water.
24. Tris-Acetate Buffer : Concentrated stock solution (50X)
Tris base - 242 g
Glacial Acetic acid - 57.1 ml
0.5 M EDTA - (pH 8.0)
Make
upto 1 litre with double distilled water.
Working stock is 1X
25. Tris
Buffer for SDS-PAGE
Tris
(Hydroxymethyl) Aminomethane
- 3.03 g
Glycine
- 14.40 g
SDS
- 1 g
Make
upto 1 litre with double distilled water.
26. T.E. buffer ( pH 8 )
10ml
Tris Cl
- (ph
8.0)
1mm
EDTA
- (pH 8.0)
10mm
Tris cl & 1mm
EDTA can be prepared from 1M Tris Cl & 0.5m
EDTA respectively.
27. 0.1
M Tris-HCl buffer pH 8.6
Tris
(Hydroxymethyl) Aminomethane
- 1.21 g
HCL
- 0.2 M (12.2ml)
NaCl
- 4g
Dissolve
in 50 ml double distilled water and adjust the volume to 200 ml.
28. Transfer Buffer (Tris
glycine buffer pH 8.3)
Tris
- 24.5 g
Glycine
- 75.0 g
Dissolve
in 10 litters of distilled water and adjust the pH.
29. Veronol
buffer 0.01 M, pH 8.6
Sodium
barbitone
- 10.00 g
Sodium
acetate
- 6. 5 g
HCl
(0.1N)
- 34.2 µl
Dissolve
in 900 ml distilled water and adjust the pH to 8.6.
Make
up the volume to 1000 ml with distilled water.
B) Enzyme substrates
1.
Starch-Iodine-Penicillin 'V' reagent
(Penicillinase substrate)
a) Iodine
reagent:
Potassium
iodide
- 1.332 g
Iodine
- 50 mg
Dissolve
in 2.5 ml distilled water and store at 40C in a brown bottle.
(Stable
for one week).
b) Penicillinase
substrate:
Soluble
starch
- 150 mg
SPB
(0.25M), pH 7.2
- 27.5 ml
Dissolve
by indirect heating. Cool the solution and add 10.64 mg penicillin 'V'
and 100 µl Iodine reagent.
2.
OPD Substrate (for peroxidase
enzyme):
Orthophenylenediamine
- 10 mg
Citrate
Phosphate Buffer pH 5.0
- 10 ml
30%
H2O2
- 10 µl
Mix
and store in a brown bottle.
3.
DAB substrate (for peroxidase
enzyme):
Diamino
benzidine
- 20 mg
Citrate
Phosphate Buffer pH 5.0
- 100 ml
30%
H2O2
- 20 µl
Mix and store in a brown bottle