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DNA Techniques Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


My DNA shoots out of the wells in my Agarose

DNA Techniques

Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.



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Old 03-26-2007, 03:45 AM
AnswerQueen AnswerQueen is offline
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Default My DNA shoots out of the wells in my Agarose

Gel... why? 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???
The tracking buffer was the same too.
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  #2 (permalink)  
Old 03-26-2007, 04:59 AM
F. J F. J is offline
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Was your tracking dye solution the same? If there is a difference in the Glycerine (at least I think that is what I used...it has been 15 years since I ran a gel) concentrations from day to day, the sample might settle differently.

Just a thought...
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Old 03-26-2007, 05:44 AM
Eugene K Eugene K is offline
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This often happens when there are traces of ethanol in your sample, as could be the case after purification using kits like PCR purification kit from Qiagen. It is extremely important to completely get rid of the washing buffer (e.g. PE in the Qiagen kit) before elution.
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Old 04-16-2008, 10:12 AM
Priyanka Priyanka is offline
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Default Re: My DNA shoots out of the wells in my Agarose

There are traces of ethanol in your samples... (you didnt dry your samples that you extracted well enough)...
All the Best
Cheers
Priyanka
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