IzziD wetlab logo
   
Search
Google
Topics
Home
How To
Reviews
Troubleshooting
What I Use
About
Subscribe in a reader
 
Other IzziDs
IzziDassorted
IzziDcode
IzziDtravel
IzziD
 
 
Site Map -- Disclaimer

What to do if you mess up and run your agarose gel backwards

Summary: If you wire your gel rig backwards causing your sample to migrate in the wrong direction, you can still recover your DNA and run your sample normally as long as the sample hasn't yet run off the edge of the gel. Simply reverse the wires to put them in the correct orientation (turn off the power supply first off course!), and run your gel normally. The samples will migrate past the loading wells and back into the correct portion of your gel. The separation should be fine, but very short bands may be slightly fainter due to diffusion.

Article created: Oct 22, 2007
Article by: Jeremiah Faith

For years I wondered what would happen if I mixed up the + and - wires when running a gel so that the DNA went in the opposite direction (remember run to red   i.e. the DNA migrates towards red terminal if you properly connect the wires to your power source). By accident I tried exactly that experiment just a few months back. I hooked the wires up to the gel rig properly, but someone had put the wires into the power source backwards (i.e. red wire into black hole and black wire into red hole). It was an essential sample and I had loaded the entire sample onto the gel; so I flipped out when I noticed that my colored loading dye was on the WRONG SIDE OF THE LOADING WELLS!!!

I immediately turned off the power supply, so that my sample wouldn't run off the edge of the gel into the vast nothingness of the TAE buffer. After a few minutes discussing my problem with the folks in the lab, the solution to try was clear: reverse the wires and hope the DNA moves back the other direction, through the loading wells, and back into the correct portion of the gel. My biggest fear with this solution was that the DNA would seep out or moved disproportionally to its length as it passed through the loading wells. Since my whole lab had wondered over the years what would happen in this situation, everyone checked in on the gel as the DNA moved seamlessly into the correct portion of the gel and migrated normally: IT WORKED!

The two major problems with this solution are: 1) if you didn't catch the problem early enough, you're sample will have run off the end of the gel and is lost (well I guess if you loaded a huge amount of DNA, you could try to recover some of it with a gigantic EtOH precipitation or a microcon type filter) and 2) your bands will certainly be a little fainter because to obtain a given amount of separation, your DNA has run further than normal (i.e. the normal distance plus 2x the distance in the wrong direction). The longer you run a gel, the more your bands diffuse and you lose DNA - particularly the shorter fragments.

Better to be careful and wire your power supply properly. But in case you were curious, I've tried it so you don't have too...