PEG precipitation of PCR products
Polyethylene glycol (PEG) precipitation, as any other PCR purification method aims to remove unincorporated primers and dNTPs from the PCR reaction so that these do not interfere in a downstream cycle sequencing reaction. Theoretically if your PCR works really well (i.e. 50+ ng of product per µl of PCR with no extra bands or primer dimers) then you can simply use 0.5 to 1.0 µl of your PCR reaction as the template without the need to clean it up before hand.
1. For a given volume of a PCR reaction, add equal amount of PEG. Mix thoroughly by vortexing or pipetting.
2. Let the PCR + PEG incubate at 37°C for 15 minutes.
3. Prepare ice cold 80% EtOH (keep in a refrigerator).
4. Centrifuge PCR + PEG at high speed (~2,500 x rcf) for 15 minutes at room temperature.
5. Remove supernatant and discard it.
6. Wash pellet with 125 µl of cold 80% EtOH added to each PCR tube.
7. Centrifuge for 2 minutes at room temperature at 1,450 rcf.
8. Remove supernatant and discard it (for plates, invert plate onto a tissue, centrifuge at 140 rcf for 1 minute).
9. Dry off the pellet to remove any traces of EtOH (visible or by smell).
10. Dissolve the pellet in equal amount of water as the original PCR reaction. Agitate lightly and let it sit for several minutes to make sure that the DNA has dissolved.
PEG Solution (20% w/v PEG, 2.5 M NaCl)
10.0 g Polyethylene glycol 8000 (MW = 6000 - 8000 is fine)
7.3 g NaCl
ddH2O up to 45 ml
Shake and let PEG go into solution. Note: PEG takes 20+ minutes to dissolve when you make the initial solution. After everything is in solution, fill with ddH2O up to 50 ml.