how to judge the result of BstXI digestion

Jose I. de las Heras jose at hgu.mrc.ac.uk
Mon Nov 19 13:44:57 EST 2001


Frank O. Fackelmayer wrote:
> 
> shude yang wrote:
> >
> > Hello, Everyone:
> >
> > I use plasmid pFL61 as a vector to built cDNA
> > library.The DNA of pFL61 has two BstXI recognise sites
> > between which there are only 20bp nucleotides.
> > Everytime I use BstXI to digeste the plasmid pFL61,I
> > can not find the difference between the moving
> > distance of digested and undigested DNA of pFL61 in
> > the agarose gel,
> 
> As undigested, supercoiled plasmid runs quite differently from
> linearized plasmid, I suspect you do not have any digestion at all when
> you DON´T see a difference in the migration, as you say.
> Did you digest at 55°C, as necessary for BstXI?
> 
> Frank

Good point. However, he won't be able to tell the difference between the
linearised (1 cut) and the completely digested (2 cuts) forms. But I
wouldn't expect many of the "1 cut only" forms, unless the digestion is
remarkably bad. Most people use much more enzyme and incubate for longer
times than is necessary. If the plasmid prep is decent and using the
right buffer, I'd personally assume that the linearised band does not
contain a significant amount of the "1 cut only" form. So yes, comparing
the mobility between the uncut and the cut plasmid is probably the
easiest way.

Jose

-- 
Dr. Jose I. de las Heras	Email: 	Jose.delasHeras at hgu.mrc.ac.uk
MRC Human Genetics Unit		Tel: 	(0131) 3322471
Chromosome Biology Section		ext: 	2121 (office)
Western General Hospital			2301 (lab)
Edinburgh EH4 2XU		Fax: 	(0131) 3432620	
Scotland - United Kingdom	

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