multiplying bands in gel purification
qiagen at kaiwan.com
Wed Feb 26 12:46:32 EST 1997
"John G. Luz" <jgluz at MAIL.MED.CORNELL.EDU> wrote in article
<330A4042.2552 at mail.med.cornell.edu>...
> 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
The extra band you observed after using the QIAGEN QIAquick Gel Extraction
kit is simply denatured (single stranded) DNA. Under certain conditions,
chaotropic reagents (present in all silica-based DNA purification methods)
can denature DNA fragments. This is a rare event that may be influenced by
sequence characteristics such as the presence of inverted repeats or
A-T-rich strectches. Because salt and buffer promote renaturation of DNA,
the following tips are recommended:
Prepare the downstream enzymatic reaction as usual, but omit the enzyme
(and any other heat-sensitive reaction components). Incubate the reaction
mix at 95°C for 2 minutes and allow the tube to cool slowly to room
temperature before adding the enzyme and proceeding.
Elute the DNA from the silica membrane with 10 mM Tris buffer containing 10
mM NaCl. the salt concentration of the eluate must then be taken into
consideration in downstream applications.
If there are any additional questions about any QIAGEN product, please feel
free to call your local QIAGEN Technical Service Department. We can be
reached in the US at 1-800-DNA-PREP from 6 am to 7 pm PST.
QIAGEN Technical Service
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