M saline physiological or ‘normal’ saline
0.14 M saline physiological or ‘normal’ saline
Sodium chloride (8.5 g/l). Store at a × 10 (for tanning erythrocytes)
0.20 M phosphate–saline buffer, pH 7.2
concentration and dilute as required. Sterilize by MATERIALS
autoclaving.
0.02 M potassium dihydrogen phosphate (KH 2 PO 4 ) (12.2 g) 0.06 M disodium hydrogen phosphate
Scintillation fluid
(Na 2 HPO 4 ) (40.4 g)
MATERIALS
0.12 M sodium chloride (36.0 g) 2,5-diphenyloxazole (PPO) (6 g)
2,2′-p-phenylene-bis (5-phenyloxazole) Dissolve in 5 l of distilled water.
METHOD
(POPOP) (0.05 g) Toluene (1000 ml)
METHOD
Polyacrylamide running buffer
Dissolve the PPO and POPOP in toluene in a fume
Stock solution cupboard. Tris–glycine buffer × 10:
TECHNICAL NOTE
144 g glycine in 1000 ml water, pH 8.3 Many institutions require the use of aqueous 30 g Tris (tris(hydroxymethyl)-aminomethane)
scintillation fluid, available ready prepared, Dilute stock solution × 10 then add 3 ml 20% SDS
although efficiency must be checked in to 600 ml of diluted stock solution.
individual systems.
A P P E N D I X A: Buffers and media
Tissue culture media
3.0 M Tris–HCl, pH 8.7
(see also information on general in vitro culture Dissolve 181.65 g Tris in 450 ml distilled water, techniques in Appendix B)
titrate to pH 8.7 with 1 M HCl, finally adjust to Many different culture media are available, each
500 ml with distilled water.
usually in an ‘old’ and ‘new’ or ‘improved’ formulation. For general use we suggest Eagle’s minimal essential medium (EMEM) and
0.1 M Tris-buffered saline, pH 8.0
Dulbecco’s modification of Eagle’s minimal
(for IgM preparation)
essential medium (DMEM) or RPMI 1640 for
MATERIALS
culture under more demanding conditions. Tris(hydroxymethyl)-aminomethane (12.1 g) Sodium chloride (29.22 g) Glycine (0.75 g)
Tris–ammonium chloride
Sodium azide (0.2 g)
(for erythrocyte lysis)
0.17 M tris(hydroxymethyl)-aminomethane 1 Dissolve ingredients in 800 ml distilled water (Tris) (20.60 g/l)
and adjust to pH 8.0 with HCl. 0.16 M ammonium chloride (8.30 g/l)
2 Make volume up to 1000 ml with distilled water.
METHOD
Add 10 ml of 0.17 M Tris to 90 ml of 0.16 M
Tris–glycine buffer, pH 8.3; 0.25 M Tris,
ammonium chloride and adjust to pH 7.2.
1.92 M glycine
This buffer induces red-cell lysis without reducing
MATERIALS
lymphocyte viability, unlike 1.0% acetic acid Tris(hydroxymethyl)-aminomethane (30.3 g) which is also used to lyse erythrocytes during
Glycine (134.6 g)
white cell counts. Sodium dodecyl sulphate (10 g)
METHOD
Dissolve materials in water and make up to
Tris–HCl buffers
1000 ml.
MATERIALS Tris(hydroxymethyl)-aminomethane (Tris)
Veronal-buffered saline
(12.1 g)
(for PEG precipitation)
Sodium chloride (85.0 g)
1 Dissolve Tris in 100 ml distilled water to prepare
Sodium barbitone (3.75 g)
a 1.0 M stock solution.
Barbitone (5.75 g)
2 Titrate to desired pH with HCl, then dilute to the required molarity.
METHOD
Dissolve and make up to 2 l with distilled water. TECHNICAL NOTE To measure and adjust the pH of a Tris solution,
This buffer is 5 times the concentration used in the it is necessary to purchase a special Calomel Tris
text, as it is more stable as a concentrated stock electrode athe electrochemistry of the normal
solution. Dilute just before use. electrode does not apply to Tris.
A P P E N D I X A: Buffers and media 351
50 M Tris
M hydrochloric acid to be added to 50 ml 0.1 10
ml 0.1 Fig. A.1 Tris buffer. Variation in
pH of Tris buffer obtained by adding 7.0 7.5 8.0 8.5 0.1 M hydrochloric acid to 50 ml
Required pH
0.1 M Tris base. Dilute the resulting mixture to the required molarity.
Carbonate Barbitone Tris Phosphate Acetate
Glycine–HCI
pH
Fig. A.2 Working ranges for useful buffers.
A P P E N D I X A: Buffers and media
APPENDIX B Basic techniques and useful data