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Reagent
Recipes Phenol prep
100 ml H20-saturated Phenol (Rathburn Chemicals)
- Add 100 ml 0.5 M Tris-Hcl pH 8.0 w/ 1mM EDTA (0.5 M TE )
- Mix and leave at room temp for 1 hr.
- Remove and discard aq. phase
- Add 100 ml 0.5 M TE, mix for 1 hr, remove aq phase and discard
- Add 100 ml 10 mM Tris-Hcl pH 8.0 w/ 1mM EDTA.
- Mix and leave at room temp for 1 hr.
- Remove aq phase and discard
- Repat (6-8) 2x => 100 ml 10 mMTE, room temp 1 hr., discard aq.
phase
- Add 100 ml 10 mM Tris-Hcl pH8
- Store at 40°C in dark. Add 0.02 % 8-hydroxyquinoline to prevent
oxidation (phenol colorless -> orange) oxidize
(Back to
the top) SOS
2% Bacto-tryptone
0.5% Bacto-yeast extract
10 mM NaCl
2.5 mM KCl
1 M sorbitol
(Back to
the top)5M
NaCl
29.0 g NaCl
Make up to 100 cm3
w/ dH20
use stir bar
Autoclave
(Back to
the top) 10%SDS
10g of Sodium dodecyl Sulphate (Sigma, high purity grade)
Make up to 100 ml w/ dH20.
Filter sterilize (0.2 µm membrane), NOT autoclaved
(Back to
the top) 5M
Potassium acetate
3M wrt Potassium and 5M wrt Acetate
RFM of 5M Kac = 5x98.15=490.75 g/dm3
To 60 mls of dH20, add 29.4 g
Add 11.5 ml glacial acetic acid
Bring up vol to 100 ml with dH 20
Stir Bar
Autoclave
(Back to
the top) 1M
Magnesium chloride
20.3 g of MgCl2·6H2O
with dH20 to 100 mL
use stir bar
sterilize by autoclaving
(Back to
the top) 0.5M
EDTA
18.6 g Disodium slat of EDTA
80ml dH20
pH with 1M NaOH or NaOH pellets to desired pH: 7.0, 7.5 or 8.0
(usually pH: 8.0)
Final volume = 100 ml
use stir bar
sterilize by autoclaving
(Back to
the top) STE
| |
500 cm3 |
200 cm3 |
100 cm3 |
| 0.1 M NaCl => |
50 cm3 |
20 cm3 |
10 cm3 |
| 10mM Tris.Cl (pH 8.0) => |
5 cm3 |
2 cm3 |
1 cm3 |
| 1mM EDTA (pH 8.0) => |
1 cm3 |
0.4 cm3 |
0.2 cm3 |
mix and autoclave |
