Description of how to prepare silica particles for the extraction of nucleic acids.
This protocol describes the preparation of Silica particles for the extraction of nucleic acids. DNA and RNA bind efficiently to acid-washed Silica particles (Boom et al., 1990).
This procedure rescues the nucleic acids from the main part of debris and interfering agents in serum and organ materials. The current method is used at DTU Vet (Oleksiewicz et al., 1998)
Principle Silica particles are sedimented twice in water, to remove the particles with a suboptimal size. Finally HCl is added to the suspension.
- This procedure involves 32% HCl Risks involved: C; R-34, R-37
- No virus is used in this procedure, however, it is essential to perform the procedure in a clean area.
- Waste treatment All waste from this procedure can be disposed of without further precautions.
- Silica particles
- Milli-Q water
- 32% HCl.
- Weigh 60 grams of Silica particles and add to a cylindrical glass tube (27.6 cm x 5 cm).
- Add Milli-Q water to a total volume of 500 ml.
- Seal with parafilm and turn the glass tube several times until Silica particles are in suspension.
- Leave to sediment 24 hours at RT.
- Pour carefully (or use suction) 440 ml supernatant off.
- Resuspend by adding Milli-Q water to 500 ml and turning the glass tube several times until Silica particles are in suspension.
- Leave to sediment for 5 hours at RT.
- Use suction to remove 440 ml supernatant. Leave 60 ml of residual volume, this will contain aproximately 50 vol% Silica. - Shake well and add 600 µl 32% HCl.
- Keep the stock in a high density polyethylene or glass bottle in the dark at RT, sealed with parafilm to reduce evaporation. This stock will keep for years.
- Make 200-500 µl aliquots in Eppendorf tubes for everyday use. Mark date of stock production.
- Vortex thoroughly before use. Is it essential that the Silica is in suspension before use.