[Methods-and-reagents] Re: sizing DNA fragments Macintosh

Michael Sullivan mlsulliv at wisc.edu
Wed Jun 29 08:57:12 EST 2005


On Jun 28, 2005, at 10:48 PM, Thomas Isenbarger wrote


A plot of log (size) vs migration is usually very linear, and in my  
experience gives very good results for sizing. When I've needed to  
carefully size bands (which is not often), I usually set up an excel  
spread sheet with the data, have it fit a straight line, and use the  
resulting equation to calculate the sizes. If you set up a template,  
it's not very cumbersome to do repeatedly with different data sets.  
Sometimes I find either the largest or smallest marker doesn't fit  
very well, so I throw it out. One think to keep in mind also is  
exactly how you measure migrations. I've heard that (and experience  
seems to bear this out) that it's most accurate to measure migration  
of the trailing edge of the band rather than the leading edge, as  
this factors out some of the effects of thick vs. thin bands.

That said, with the availability of relatively inexpensive calibrated  
markers, I find it pretty rare that I actually need to go through  
this, and can usually get adequate estimates of sizes by visual  
examination. What are you doing that you want to calculate band sizes  
more accurately?

Hope this helps.

Mike


---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)


>
> thanks for the help, Kevin.
>
> but, i have tried this, and i am looking for a quicker solution.
>
> 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.
>
> 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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