IUBio

Accuracy of electrophoresis

Ben Mijts ben at trishul.sci.gu.edu.au
Wed Mar 31 23:36:33 EST 1999


It certainly isn't possible to distinguish 100000 and 100001 bp DNA on a pulsed
field gel system. Depending on the gel conditions (switch time, temperature,
agarose purity, etc, etc, etc...) you may be able to just distinguish fragments
a couple of hunderd base pairs different in size. Very generally speaking gel
resolution is relative to the size of the fragments being separated. If you
wish to resolve frgaments 1 bp different in size you would be limited to
relatively small DNA fragments using a technique such as denaturing gradient
gel electrophoresis.

As for determining gel accuracy it all depends on how you are reading the bands
in the gel, remember this that DNA is visualised under UV light and that even
under the best conditions there will be variations. Using sophisticated
software in order to convert the bands into discrete data would be needed to
properly characterize gel accuracy.

Basically I'm saying gel electrophoresis isn't a particularly accurate
technique under the best conditions and considerable error in results is
unavoidable.



Zasha Weinberg wrote:

> I'm trying to find out information on how accurate gel electrophoresis and
> pulsed field electrophoresis are.   As may be clear, my expertise is not in
> biology (it's in computer science).
>
> For example, suppose I have a number of oligonucleotides, and wish to
> identify the one of length 100000 bases (for the pulsed field case).  Will
> it be accurate enough to distinguish that from one of length=100001.  In
> general, is it possible to characterize the accuracy, assuming I want to be
> as accurate as possible?  Also, what kind of lab time would this require?
>
> Thanks,
>
> -Z.




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