multiplying bands in gel purification
seppen at u.washington.edu
Thu Feb 20 14:43:49 EST 1997
On 18 Feb 1997, John G. Luz wrote:
> Folks--I'm tearing my hair out. I cut a nice clean tight band out of a
> gel, and purified it with the qiagen gel extraction kit. But when I ran
> the gel-purified band out on a gel to check my yield, I got two bands,
> both about equimolar. One band looked about the right size. The other
> looked about 1kb smaller. The band I gel-purified was 5.4 kb and cut
> with NdeI and Xho. What's going on?--John
I have observed a similar phenomenon many times. Some inserts run at a
smaller molecular size after isolation. First this worried me a lot, but
when I simply ignored it and went on with a ligation, the insert in the
new vector had the correct size...
My explanation; Some pieces of DNA have a tendency to form a secondary
structure which makes them run faster on an agarose gel. This secondary
structure preferably forms after gel isolation. Because the DNA is
cleaner, contaminants from the isolation kit, who knows.
My advise therefore would be, ignore and continue.
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