Materials :
• calf thymus DNA - DNase I - Phenol - Chloroform - Liquid nitrogen - Ethanol- TE buffer
Solutions:
- prepare 10 mL of dilution buffer (20 mM Tris-HCl pH 8.0, 10 mM MgCl2)
| |
Use stock |
Dilute |
for 6.5 mL (final volume: 10 mL) |
| Tris HCl pH 8.0 |
1 M |
1/50 |
200 µL |
| MgCl2 |
1 M (4 g for 20 mL) |
1/100 |
100 µL |
| H2O |
|
|
6.2 mL |
- 3 M sodium acetate: 40.8 g for 100 mL H2O
- 0.5 M EDTA
- 10 % SDS
- DNase I: 10 mg/mL in water
- phenol/chloroform (1:1)
Protocol description:
DNase I hydrolysis
- use 3.5 mL of calf thymus DNA at 10 mg/mL (weight 35 mg of DNA)
- add 6.5 mL of dilution buffer
- add 50 µL of DNase I
- mix a few seconds until reduced viscosity
- freeze in liquid nitrogen
- add 1 mL 0.5 M EDTA
- allow melting
- add 1 mL of 10 % SDS
Purification
- add 10 mL of phenol/chloroform ; vortex thoroughly
- centrifuge 5 min at 12,000 rpm (Beckman JA 12 rotor)
- take the aqueous phase
- add 10 mL of phenol/chloroform ; vortex thoroughly
- centrifuge 5 min at 12,000 rpm
- take the aqueous phase
- add 1 mL of 3 M sodium acetate
- add 20 mL of ethanol ; mix gently
- allow precipitation by keeping in the ice
- centrifuge 5 min at 12,000 rpm
- wash the pellet with ethanol ; let it dry
- resuspend the dry pellet with 3.5 mL of TE buffer
- store at 4°C
- measure absorbance at 260 nm
- control the heterogeneity of the cleaved DNA by electrophoresis on a 2% agarose gel
References: unpublished procedure
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