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My DNA shoots out of the wells in my Agarose Gel... why? - Agarose Gel Loading Help (Jan/29/2007 )

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I have DNA that I'm cutting with BamH1 and Pml1. They use different buffers so I do a PCR purification in between each digestion. After an hour each of digest, I add the correct amount of loading buffer to my samples and load my 1.3% agarose gel in 1X TAE buffer. After loading the gel, my samples quickly disperse out of the well. The wells are definately deep enough. It's like the sample is "running away" from the agarose. Earlier today I used the SAME running buffer, and SAME agarose and the SAME box to run some samples that worked just fine. This happened to me before with BamH1 and another restriction enzyme. Do you know what's going on???


you have ethanol in your samples. most likely left over from the purification step.



I agree. residue ethanol is the cause.


If you are sure that don't have ethanol then check that the loading buffer has enough glycerol. some times 20% glycerol isnot enough to retain tha sample
I prefer to prepare 30%glycerol so I'm sure it will sink to the bottom of the well. or if you are using sucrose or ficoll to give the weigh to the sample could be also the problem.


The same thing happened to me today. Tomorrow I'm changing protocol ommiting purification (and risking protein contamination) after digestion to see if EtOH really is troublemaker. M.


DNA still shooting out mad.gif. My buffer has ~30% glycerol... M.


QUOTE (Frozen @ Mar 14 2007, 10:22 AM)
DNA still shooting out mad.gif. My buffer has ~30% glycerol... M.

maybe you are pipetting too fast.


Maybe you can try to wait a bit longer for ethanol to evaporate during your purification steps...


Maybe you are witnessing antigravity. wink.gif


are your wells too shallow? how thick is the gel? how deep the wells? are you pipetting abruptly?


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