* Denaturing solution
4 M Guanidine isothiocyanate
10 mM Tris-HCl (pH 7.0)
1 mM EDTA
0.5% 2-mercaptoethanol
Denaturing solution: 10 ml in total
Guanidine isothiocyanate (MW 118.16)
4.73 g
1M Tris-HCl (pH 7.0)
100 μl
0.5M EDTA
20 μl
Add water to 9.95 ml in total.
Autoclave.
Add 50 μl 2-mercaptoethanol just before use. (5 μl of 2-mercaptoethanol per 1 ml Denaturing Solution)
Note: Keep the Denaturing solution at 4 °C. Do not use buffer older than one week. If possible, make fresh buffer to use.
**Extraction Buffer
100 mM Sodium phosphate buffer [pH 7.0]
100 mM Tris-HCl [pH 7.0]
100 mM EDTA [pH 8.0]
1.5 M NaCl
1% Hexadecyltrimethylammonium bromide (CTAB)
2% SDS
Extraction Buffer for 1 L in total
1M Sodium phosphate buffer [pH 7.0]*
100 ml
1M Tris-HCl [pH 7.0]
100 ml
0.5M EDTA [pH 8.0]
200 ml
5 M NaCl
300 ml
Autoclave and keep it at room temperature.
Add 200ml, 5% Hexadecyltrimethylammonium bromide (CTAB, autoclaved) and 100 ml, 20% SDS (autoclaved) just before use. If CTAB was crystallized, melt it at 60 °C.
* 1M Sodium phosphate buffer: 57.7 ml, 1M Sodium phosphate monobasic (NaH2PO4) and 42.3 ml, 1M Sodium phosphate dibasic (NaH2PO4). Adjust pH to 7.0
Note: Do not use 20 % SDS if it has precipitation. It is normal to see milky suspension when you add SDS to the solution. Once you add SDS, place the extraction buffer at 60 °C to ensure SDS is well suspended.