Size selective DNA precipitation protocol
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Solutions/reagents:
- <a name="DNA sample">DNA sample
<tab>(DNA to be separated (e.g. PCR reaction mixture))</a> - <a name="PEG/MgCl2">PEG/MgCl2
<tab>(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))</a> - <a name="TE buffer">TE buffer
<tab>(10 mM TRIS-HCl, 1 mM EDTA, pH 8.0)</a> - buffer of choice
Equipment:
- Centrifuge
- Eppendorf tubes
Steps:
- Measure out 50 µl of <a href="#DNA sample" >DNA sample</a> into Eppendorf tube (1).
Measure out 150 µl of <a href="#TE buffer" >TE buffer</a> into DNA sample.
Gently tap the mixture for a few secs. - Measure out 10 µl of <a href="#PEG/MgCl2" >PEG/MgCl2</a> 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
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