Size selective DNA precipitation protocol
- DNA sample (DNA to be separated (e.g. PCR reaction mixture))
- PEG/MgCl2 (30% (w/v) PEG 8000/30 mM MgCl2 (concentration of PEG 8000 can be varied to shift the size of the percipitated DNA. The concentration used here will remove DNA fragments with less than 300bp))
- TE buffer (10 mM TRIS-HCl, 1 mM EDTA, pH 8.0)
- buffer of choice
- Eppendorf tubes
- Measure out 50 µl of DNA sample into Eppendorf tube (1).
Measure out 150 µl of TE buffer into DNA sample.
Gently tap the mixture for a few secs.
- Measure out 10 µl of PEG/MgCl2 into Eppendorf tube (1).
- Vortex the mixture for a few secs.
- Centrifuge at a speed of 10000 Xg for 15 mins at room temperature, gently aspirate out the supernatant and discard it.
- Carefully remove supernatant not to disturb the pellet, which will be invisible.
- Add buffer of choice to pellet.
Add appropriate volume of buffer.
Dissolve the pellet in the solution.
TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 15 mins