small DNA fragment recovery

m-brazil at nimr.mrc.ac.uk m-brazil at nimr.mrc.ac.uk
Sat Oct 1 07:08:14 EST 1994


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> From server-daemon at dl.ac.uk Sat Oct  1 00:39:16 1994
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> From: salger at wap18.zi.biologie.uni-muenchen.de (Klaus Salger)
> Reply-To: salger at wap18.zi.biologie.uni-muenchen.de (Klaus Salger)



> 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
> 
> 
> 


Dear Klaus,

This sounds like a great method for isolating DNA fragments. Have you used
it on larger fragments, eg 5kb? 

Also, do you place you tubes containing the melted agarose /DNA, in an
isopropanol bath, in a -70 freezer?

Thanks
Melanie
(m-brazil at uk.ac.mrc.nimr)




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