Agarose-free ‘gel excision’

Agarose-free ‘gel excision’

Sometimes the best way to avoid something is to … avoid it?

Thermofisher has something called clone-well. It is a product that has two sets of wells, uses Sybr Safe for efficient blue-light illumination and is expensive (~ $20 CA per gel as of writing).
The principle behind clone-well is simple. You load DNA in the wells closest to the black electrode and collect DNA from a second set of wells closest to the red electrode.
If you have a bluelight transilluminator such as the ‘Dark Reader’ and regularly make gels you’re set to duplicate the functionality of clone-well gels inexpensively.

Cast a gel with two sets of wells.

Here is a cartoon of the gel you’ll set up.
This gel will be 0.5–0.8% agarose. I use wide, thin  wells. Use 2X SybrSafe dye or equivalent. The gel will be in your gel box. Before loading the gel remember to place the gel box complete with gel and buffer ontop of the blue light transilluminator.
Load your DNA as per usual. Run the gel more slowly. With lithium borate/sodium borate electrophoresis buffers I use ~ 150–200 V for a 10 cm gel.
Once the lower dye front is in between points A and B (gel cartoon above), turn on the blue light and observe the position of your band. Because your gel rig will hoist the gel a few cm from direct contact with the blue light transilluminator, the extra SybrSafe dye comes in handy.
Check on the migration of the DNA. Once it arrives at point B use a 200 uL pipette to remove the DNA/buffer mixture from the wells at B.
The DNA collected in this way works for PCR with NEB Q5 and works well with the NEB Gibson HiFi Mix (I tried a three fragment assembly).