PEG-3350 PRECIPITATION

For purifying PCR products larger than 300 bp.

1) Move aqueous portion of PCR products to a new, labelled 1.5 ml tube.

2) Add 1/2 the aqueous volume of 30% PEG 3350 (in 1.5M NaCl) to your PCR reaction (e.g. add 25 ul of PEG solution to a 50 ul PCR reaction).

3) Vortex gently and let sit for 10-15 min at room temperature.

4) Centrifuge 8 min at max RPM (14K).  PCR products larger than 300 bp will pellet.  PCR products smaller than 300 bp (e.g. dNTPs, PCR primers) will remain in the supernatant.

5) Carefully remove the supernatant with a pipet without disturbing the invisible pellet.

6) Gently (no mixing) add 300 ul 75% EtOH to rinse the PEG pellet and then spin 4 min at max RPM (14K).  Carefully pour off the EtOH while being careful to avoid disturbing the pellet

7) Repeat step 5 (OPTIONAL).

8) Dry the remaining EtOH from the pellet on the Speedvac under vacuum, with medium heat (about 15 min).

9) Resuspend the pellet (DNA) in a small volume (~12-25 ul) of 10 mM Tris and place in 37C incubator for 15 minutes or until resuspended.  With experience, you will get a feel for your % recoveries from PEG precipitation, and a better gauge on resuspension volumes.  This volume may also be dependent on the how successful the initial PCR reaction was.

9) Run 4-5 ul on an agarose gel to assess recovery (OPTIONAL).