[Methods-and-reagents] Re: sizing DNA fragments Macintosh
Austin P. So (Hae Jin)
nobody at nowhere.com
Tue Jun 28 23:51:36 EST 2005
Thomas Isenbarger wrote:
> In article <d9suej$fa3d$1 at netnews.upenn.edu>,
> kjude at mail2.sas.upenn.edu (Kevin M Jude) wrote:
>>It can be done with any plotting software; prepare a standard curve and
>>see where your bands fall. Migration rate increases logarithmically with
> but, i have tried this, and i am looking for a quicker solution.
I don't think there is a "quick" solution...unless you are absolutely
sure your buffer composition, your gel percentage, your current, and
your temperature of running conditions...DNA will even "smile" across a
gel, so that migration distances are not uniform...
> i plotted the data in igor, then fit 1/distance versus size with a
> second order polynomial. then i used the fit equation and the distances
> of the unknowns to solve the quadratic equation for the sizes of the
> unknowns. . .and my control plasmid band was 0.5 Kbp off.
Your mistake was plotting 1/distance. You are introducing a big error in
A reciprocal plot is nowhere near the same as a logarithm fit BTW...why
on earth did you do this?
*Always* fit to the raw data. And make sure your standards fall within
the resolution capabilities of the gel and that your unknowns fall
within the range of your standard curve.
> so, i am looking for a quicker method with better fitting to do this.
> there are published fitting algorithms that are better than a simple log
> fit, so i am hoping to find software that has these implemented.
> when you say "see where your bands fall", are you saying just eyeball
> the place on the curve where my unknowns fall?
> this is a bit too rough for me.
> any other help?
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