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Old 11-05-2012, 03:47 AM   #2
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
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I can only speculate. Maybe the agarose gel was run either without EtBr, then post run stained, or no EtBr was put in the gel or loading solution, but was put in the buffer? In either of these cases, the products would migrate mostly or entirely in the absence of EtBr.

Why is that important? Well, I have not tested this myself, but Dave Cook at Sage Science told me that in the absence of EtBr (or other DNA binding dyes) the difference in mobility between fully double-stranded amplicons, and "bubble products" (double stranded only at the adapter ends) does not happen. I.e. everything basically runs at the "correct" size.

So, if this is the issue you see, your amplicons are fine, but consist of variable amounts of normal and bubble-products. The Bioanalyzer chip faithfully displays the difference with the bubble products migrating slower than their true molecular weight.

Again, just a guess.

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Phillip
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