Library insert DNA

jwobrien jwobrien at students.wisc.edu
Mon Nov 25 14:07:02 EST 1996


In article <56vh97$dr9 at mserv1.dl.ac.uk>, robrien at ollamh.ucd.ie wrote:

> Hi All,
> I would like some advice on the best way of preparing DNA (mycobacterial)
> for making a total genomic library, ie: randomly sheared, 4-6kb 
> fragments suitable for cloning into a suitable plasmid. I have been 
> preparing insert DNA by partial digestion with Mbo1 followed by gel 
> electrophoresis to cut out the correct size fragments in low-melt 
> gel. I have then extracted the melted gel slice with phenol and 
> recovered the DNA by ethanol precipitation. (There has been too high a gel 
> volume, 7-10 mls, to put it through a wizard column or similar).
> Trouble is, the insert DNA made in this way has been extremely 
> reluctant to ligate into my vector (pGEM, CIPed with phosphatase). 
> The vector DNA does not appear to be the problem as it will ligate
> generic Sau3A fragments very well. I am concerned that the phenol 
> treatment may be nibbling back on my ends, even though it is high 
> grade, redistilled. Does anybody have a gentle method of recovering 
> fragments from a largish gel volume ?
> Advice, tips and time-tested methods would be very much appreciated !
> 
> -Rory
> 
> Rory O'Brien.
> National Agricultural and Veterinary Biotechnology Centre,
> University College Dublin,
> Belfield, Dublin 4.
> IRELAND
> 
> Ph: +353 (1) 7062815
> Fax:+353 (1) 2692016
> e-mail: robrien at ollamh.ucd.ie

I have successfully made libraries from several dilfferent bacteria using
essentially the method you described. The only differences were 1) I used
Sau3AI for the partials (that shouldn't matter) and 2) after gel
purificaton I used Gelase to digest the lmp agarose instead of phenol
(that may or may not matter).  My guess is that the problem you are having
may be that, when you excise the fragments from the preparative gel, you
are exposing them to UV light. If this is true, even a few seconds of
exposure can cause enough damage to the DNA to dramatically reduce its
effectiveness in transforming the host. 

If you are exposing the partials to UV, it is a good idea to avoid
exposure by using guide lanes. In other words, try running a gel with mw
markers in one lane, a small amount of your partials in a second lane and
the majority of your partials in a third lane. Cut out and stain the first
two lanes, expose them to UV, mark the region of the gel with the sizes
you want and use them as a guide to find the sizes of partials you are
looking for in the third lane. This way the partials never get exposed. 

If you aren't exposing the fragments, I don't know what the problem is. It
is possible it relates to methylation of the input DNA vs the restriction
systems of the host. Good luck. 

Tom Schoenfeld, Senior Scientist
Epicentre Technologies, Inc.
1202 Ann Street 
Madison, WI 53713 USA
ph. 608-258-3090
fax 608-258-3099
e-mail tschoenf at unix.midplains.net



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