??IBI-UEA electroelutor

Ted Michelini Tedm at darkwing.uoregon.edu
Mon Apr 24 13:54:59 EST 1995


In article <3n6ia8$i9r at uwm.edu>, lkelly at alpha1.csd.uwm.edu (Louise Ann
Kelly) wrote:

> Hi all,
> 
> I'm looking for hints on optimizing recovery of 400-900bp oligos with this
> instrument. The manufacturer suggests 90% recovery, but ours is ~25-30%.
> The protocol involves cutting bands from agarose, and electophesing into
> V-channel that contains high-salt buffer with marker. Fragments are
> recovered by standard ethanol precipitation. 

> We're going to try 10-13M NH4OAc next. Any advice in this matter will be
> appreciated.  We do use gene clean, but we're low on funds; optimizing this
> thing would be a blessing. The manufacturer was helpful, but they didn't
> have anyone around that used it either.
> 
> Please reply by e-mail, lkelly at uwm.csd.edu or post.
> 
> Thanks, Luanne Kelly


Luanne,
      13M NH4OAc?  UGH!  In my experience all the fancy apparati sold to
recover DNA from gels quote "quatitative" yields and DO NOT DELIVER. I
have tried many of them, including the IBI- Mr. Salty, and a Amicon unit
which ultrafilters along with electro-elution. They all require great
chemical and physical manipulation of your DNA, which takes a lot of
time.  Now in some cases you may need the quantitative yields these units
claim, if so good luck.  If however you are cloning then the yields from
vastly simple and cheap methods are good enough and the DNA is very clean
and unperturbed. 
   The best method I have found is the Millex spin filters from Millipore,
a simple excised gel band, a freeze thaw cycle and a short spin, sometimes
with a warm TE wash,  and you have your 40-60% yield sitting in nice mild
TAE or TBE. A thorough EtOH precip, with or without glycogen, and you're
ready to go. Other filter units are similar, but can't take the freeze
thaw cycle, or a full hard spin. There is even a method from Biotechniques
which uses a 30 cent syringe to squeeze the DNA out. Gene clean etc mangle
the DNA a little more but are fast and work well for multiple samples.
Luanne, if your really low on funds I hope someone can help you get your
yields up on the IBI, but I suspect it isn't a trivial task.. Good Luck.
                                                   Ted M
                                                   Tedm at darkwing.uoregon.edu



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