Dear all,

bugg at mbcf.stjude.org bugg at mbcf.stjude.org
Fri Nov 15 10:38:17 EST 1991


In article <10247.9111130031 at crc.ac.uk>, kraj%uk.ac.crc at VTVM2.CC.VT.EDU (Mr. K.R. Raj) writes:
> 
>                 I am quite tired of electroeluting DNA out of agarose. Could
>  anyony
>              anyone suggest a better way other than using the geneclean kit ?
>                    Thankyou.
>                                                 kraj at uk.ac.crc
-- 
In your posting, you requested information about other methods to elute DNA
from agarose besides electroelution and Gene-clean (glass milk?).  
Do you really need to elute the DNA?  For example, if you use one of FMC's 
high quality low melt agaroses such as SeaPlaque or NuSieve, you can perform
ligation, restriction digestion, labelling, etc. with the gel present.
However, if you really do need to get rid of the agarose, here are two methods
that I use.
First of all, for both of these methods, you still need to use SeaPlaque or
NuSieve (or some other high-quality, low melting point agarose) and you need
to run the gel in Tris-acetate-EDTA buffer (not Tris-borate-EDTA).
1.  Warm phenol method - After slicing out your band from the gel, add 100 ul
of sterile distilled water per lane, and melt at 65C for 10 min.  At the same
time, heat some Tris-saturated phenol in a separate tube in
the 65C bath.  Add an equal volume of warm phenol to the DNA-agarose and 
vortex vigorously.  Microfuge to separate phases and remove the aqueous phase
to a fresh tube.  Add another equal volume of TE to the organic phase and heat
at 65C again for 10 more minutes.  Vortex as before and separate phases. 
Combine the two aqueous phases and ether extract.  Then, precipitate the DNA
with 2 volumes of cold (-20C) EtOH.  You don't have to add any salt.  Leave on
ice for 10 min and microfuge 15 min.  Wash the pellet with 70% EtOH and dry. 
Resuspend in TE or dH2O.  This DNA can be used for ligation, digestion,
labelling, etc.
2.  Agarase digestion - Use one of the commercially available agarases to
digest the agarose present in the slice and then just EtOH precipitate the DNA
without any extractions.  Both New England Biolabs and Epicentre Technologies
have agarases available.  Epicentre calls theirs "GELase".  I have used GELase
many times with good results.  It is especially nice if you have to do a second
restriction digest and then run a second gel.  This is hard to do with the
agarose present because it has to remain hot while you load it on the second
gel!
Good luck!
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