APPENDIX 2 - REAGENTS
0.5 M EDTA pH 8.0
To ~300 mL of distilled water in a beaker, add 93.05 g EDTA
Add ~10 g NaOH pellets
Stir until dissolved
Adjust pH to 8.0 with 10 M NaOH
Adjust volume to 500 mL with distilled H2O
Autoclave to sterilise
2 M Tris pH 7.4
To ~850 mL of distilled water in a beaker, add 242.2 g Tris Base
Add 75 mL concentrated HCl
Add concentrated HCl slowly and carefully, with stirring, until
pH is 7.4
Adjust volume to 1000 mL with distilled water
Autoclave to sterilise
1M Tris-HCl pH 8.2
To ~400 mL of distilled water in a beaker, add 78.8 g Tris-HCl
Adjust pH to 8.2
Adjust volume to 500 mL with distilled water
Autoclave to sterilise
Cell Lysis Buffer pH 7.5
(20 mM Tris, 5 mM EDTA)
10 mL 2 M Tris stock
10 mL 0.5 M EDTA stock
Adjust volume to 1000 mL with distilled water
Salting Out Digestion Buffer pH 8.2
(20 mM Tris-HCl, 5 mM EDTA, 400 mM NaCl)
20 mL 1 M Tris-HCl stock
10 mL 0.5 M EDTA stock
23.38 g NaCl
Adjust volume to 1000 mL with distilled water
TE Buffer
(10 mM Tris, pH 7.4; 0.1 mM EDTA, pH 8.0)
0.5 mL stock 2 M Tris
20 mL stock 0.5 M EDTA
Adjust volume to 100 mL with distilled water
50X TAE
24.2 g Tris base
5.71 mL glacial acetic acid
10 mL 0.5 M EDTA (pH 8.0)
Adjust volume to 100 mL with distilled water
5X TBE
54 g Tris base
27.5 g boric acid
20 mL 0.5 M EDTA (pH 8.0)
Adjust volume to 1 L with distilled water
10% SDS
20 g SDS powder
Adjust volume to 200 mL with distilled water
**Avoid breathing dust** **Do not autoclave**
6X Gel-loading Buffer
0.25% bromophenol blue
0.25% xylene cyanol FF
30% gycerol in water
Ethidium Bromide Staining Bath
(2 mg/L)
0.1 mL stock 5 mg/mL ethidium bromide
Adjust volume to 250 mL with distilled water
20% APS
2 g ammonium persulphate
Adjust volume to 10 mL with distilled water
10 M NaOH
To ~300 mL distilled water in a beaker, add 200 g of NaOH pellets
Adjust volume to 500 mL with distilled water
**Handle NaOH carefully**
Saturated NaCl (~6 M)
175 g NaCl
Adjust volume to 500 mL with distilled water
10 mg/mL Proteinase K
100 mg proteinase K
Adjust volume to 10 mL with sterile distilled water
10 mM dNTP's
(10 mM dNTP's, 100 mL)
10 mL 100 mM dATP
10 mL 100 mM dGTP
10 mL 100 mM dCTP
10 mL 100 mM dTTP
60 mL sterile distilled water
2% Agarose
4 g electrophoresis grade agarose
4 mL stock 50X TAE
Adjust volume to 200 mL with distilled water
Heat until all agarose has dissolved
Use 10 mL for each 5 cm x 7.5 cm plate
30% Acrylamide
116 g acrylamide
4 g bis-acrylamide
Adjust volume to 400 mL with distilled water
Heat solution to 37C to dissolve
5% Acrylamide Gels
10 mL 5X TBE
8.3 mL 30% acrylamide
31.35 mL distilled water
350 mL APS
50 mL TEMED
8% Acrylamide Gels
10 mL 5X TBE
13.3 mL 30% acrylamide
27.7 mL distilled water
350 mL APS
50 mL TEMED