small DNA fragment recovery

Klaus Salger salger at wap18.zi.biologie.uni-muenchen.de
Fri Sep 30 17:30:18 EST 1994


David B. Stern (ds28 at cornell.edu) wrote:
: What is the best way to get high yields of small DNA fragments (50-100bp)
: isolated from agarose gels?
: I have tried Qiagen and other glass products, but my yields always seem way
: low.
: Do freeze squeeze, phenol extractions or Gelase type products work as well?
: Thanks
: phk1 at cornell.edu
: Phil Kogan

David, I would recommend Jim Graham's method. I used it several times
with small fragments (100-150bp). It's fast and gives high yields.
I used the fragments for labling or cloning - no problem.
The following is taken from Paul Hengen's
         *************************************************************
         *           Frequently Asked Question (FAQ) list            *
         *            for bionet.molbio.methds-reagnts               *
         *       version 01.23.08.1994 (version.day.month.year)      *
         *************************************************************
which is available via anonymous FTP from ftp.ncifcrf.gov as file
pub/methods/FAQlist or upon request by e-mail to pnh at ncifcrf.gov :

Low-melt agarose method from Jim Graham (jgraham at bronze.ucs.indiana.edu):

Cut your vector and insert fragments and run them out on a low melting agarose
gel (eg. SeaKem) in TAE buffer.  Excise the bands of interest on a LONG WAVE UV
box making sure to cut the smallest slice possible.  For very low percentage
agarose, use a thin layer of standard agaorse (1%) as a support by pouring it
without a comb, setting the comb higher, and pouring your low melting agarose
gel on top once the support layer has set.  Cut this layer off as well when you
excise your bands.  Place  the gel slices in sterile eppendorfs and melt them
at 70C in a beaker. Do not add any buffer or dilute the slices. Carry this
beaker to the -70 freezer and put the samples in an isopropanol bath. Wait 5
minutes, and then thaw them.  Spin 5 minutes in the microfuge. The supenatant
is your sample for ligation.
<snip>

Cheers
  Klaus


Klaus Salger
Zoologisches Institut
AG MacWilliams
Luisenstr. 14
80333 Muenchen
Germany

phone : ++49 (0)89 5902 -502
FAX   :                 -450
e-mail: salger at zi.biologie.uni-muenchen.de



More information about the Methods mailing list