Extraction of DNA from agarose gels

[Clemens Suter-Crazzolara]

> 
> In article <36em01$5p at emory.mathcs.emory.edu>, medtjm at bimcore.emory.edu (T. J. Murphy) writes:
> >In article 15518 at leeds.ac.uk, futers at biovax.leeds.ac.uk writes:
> >> In article <GABRE.40.2E87E76D at wwg3.uovs.ac.za>, GABRE at wwg3.uovs.ac.za writes:
> >> >Hi,
> >> >
> >> >We are currently looking for an efficient and fast method for the isolation 
> >> >of DNA fragments from agarose gels.> >
> >> 
> >> The quickest method I know is as follows:-
> >>     1. Cut band out of agarose gel.
> >>     2. Put into 1.5ml tupe and freeze in liquid nitrogen.
> >>     3. Place frozen agarose slice onto a piece of Nescofilm.
> >>     4. Squeeze between finger and thumb and as the liquid is
> >>        squeezed out, collect with a pipettor.
> >> 
> >>   You are left with only a small amount of agarose in the Nescofilm
> >> and you should get a high recovery of the DNA which can be used 
> >> directly in ligations etc.
> >>            Hope this helps,
> >>                   Simon Futers (futers at biovax.leeds.ac.uk)
> >> 
> >> 
> >
> >What is the best agarose to use for these kinds of crush protocols????
> >
> >
> >TJ Murphy
> >Asst. Professor	      
> >Dept of Pharmacology
> >Emory University School of Medicine   	    
> >
> >
> 
> I have always used "ordinary" agarose.  I do not think there is any best
> agarose for this technique.
>                      Simon Futers (futers at biovax.leeds.ac.uk)
> 
> 
> 
> 
not so ! I guess that by coincedence you have been using a good
agarose, like f.i. the once from FMC. Agarose may contain a number
of contaminants that screw up digestions and ligations. IMHO the
agarose quality is essential for enzymatic reactions. So if you
isolate lambda DNA from lysed plates, don't use agar, but a high
quality agarose. Also, using agarAse to remove your agarOse is useles,
if the latter is crappy.
Which brings me to another point: is it really essential to isolate
DNA fragments from your gel pieces ? I usually ligate in low melting
point agarose itself, without purification. For multiprime labelling,
I also use LMA. efficiencies are naturally a little lower for transfactions,
but if you need only a couple of clones.....

clemens, heidelberg