Desalting superquick protocol

Sometime ligation or recombination reaction (BP or LR) contain to much salt for electrocompetent cells which causes the cells to explode during electroporation. To achieve good transformation efficiency the salt concentration must be decreased either by reducing the volume of the ligation reaction used in the transformation or by diluting down the reaction; both will yield less transformants…

 

Most DNA desalting or buffer change procedures involve salt and alcohol (or isopropanol) precipitation. Oftentimes precipitation is performed at -20C, and then high speed centrifugation in a benchtop refrigerated centrifuge is necessary, followed by a couple pellet washes in 70% ethanol. The following is a super quick procedure which I got from Marie-Josée Morency a biologist in my lab. From start to finish it take 30 minutes, and is based on dialysis rather than precipitation. The procedure is effective, fast, cheap (roughly 60 cent to 1.25$/dialysis) and very robust and works 100% of the times.

 

Procedure :

 

  1. Place roughly 25ml of Nanopure (miliQ, dd) water in a Petri.
  2. Take a MF-Millipore Membrane (catalog number VSWP02500), cut it in half*.
  3. Carefully place the membrane on the water in the petri.
  4. Carefully place the your ligation or recombination reaction on the membrane (see picture).
  5. Wait 30 minutes.
  6. Recover.

 

*If cost is an issue the membrane can be cut in four. The petri is reusable of course.

Top view of the drop
Top view of the drop
Top view of the drop
Top view of the drop
Side view through the petri wall
Side view through the petri wall