Don't let your DNA sample float away when loading an agarose gel

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Article created: Oct 26, 2007
Article by: Jeremiah Faith

There have been a few unfortunate and seemingly random events in my wetlab life where I loaded an agarose gel with my sample, only to see the sample diffuse extremely quickly out of the gel well and into the surrounding buffer. This phenomenon can be a horrifying event, because it seems to occur at random, you never know when it my strike and ruin your experiment. Thankfully this disappearing sample trick has rarely occurred with my samples, but it occurred often enough that I decided to investigate the problem.

My sample had a much higher chance of disappearing when I cleaned it using a Qiagen PCR purification kit, particularly when I had loaded the entire 30 ul elution volume into a gel rather than diluting the purified DNA into TE or some other solution. I also noticed that the problem never occurred when I was loading a gel with salty solutions like restriction digests. I decided it was either something to do with the EB buffer supplied by Qiagen, the salt concentration of my sample relative to that of TAE, or residual amounts of ethanol (EtOH) that were eluted with the EB on the final step of the Qiagen purification.

Click image for larger

I ran a gel to test these ideas. My base sample was 1.5 ul of 25 bp ladder (Invitrogen Part No: 10597-011) with 2 ul of 5x sucrose loading dye. I dye + ladder into 10 ul of either EB, EB + 15%EtOH, TE, or STE (TE with 50 mM salt). I also included a NEB 2-log ladder as a standard (Part No: N3200S). To my disappointment, none of the samples shot out of the well in the characteristic quick manner I was hoping to see. However, the EtOH sample did seem to leak out a little. When I looked at the results on an agarose gel (see the Figure on the right), it was clear that the sample with EtOH had largely diffused away (second lane from the left).

Many months (years?) later, I had a second confirmation of this problem when I stumbled upon Frequently Asked Question Number 205 on Qiagen's website (and yes, I had looked there before and scavenged the internet to solve this problem in the past and never found anything).

FAQ 205: Why does my DNA sample float out of the slot when loading it into an agarose gel?

Answer: DNA fragments purified with the QIAGEN DNA Cleanup Systems, i.e., the QIAquick PCR Purification Kit, the MinElute Reaction Cleanup Kit, the QIAEX II Gel Extraction Kit etc. may float out of the loading wells of agarose gels due to residual ethanol carried over from the wash step with Buffer PE (despite the addition of glycerol-containing loading buffer).

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Don't let your DNA sample float away when loading an agarose gel