Library construction -- mechanical shearing

Chris Boyd chrisb at hgu.mrc.ac.uk
Tue Dec 2 05:50:04 EST 1997


Shaun Tyler (styler at HWC.CA) wrote:
: I=92m interested in sequencing a 40 kb plasmid (a native one not a clone)
: for which I have a limited amount of material and no restriction map.=20
: Since what I have was hard to come by I=92m hesitant to waste it in
: establishing a restriction map in order to subclone more manageable
: fragments.  Our sequencing capabilities are not a limiting factor so I
: was thinking of using a shotgun approach with randomly sheared material
: for a library and then closing the gaps by PCR and primer walking.=20
: Unfortunately I have been unable to find any protocols for preparing
: mechanically sheared DNA.  I know people occasionally use this method
: for preparing genomic libraries but I=92m not sure how effective this
: would be on something relatively small like a 40 kb plasmid.  Any input
: would be greatly appreciated.

Paul Hengen (what would we do without him?) surveyed this in TIBS
(1997) 22, 273-274.

A nebulizing approach cited therein can be found close to you at
http://megasun.bch.umontreal.ca/ogmp/bank.html

You'll still need to verify the sequence in the end by a certain amount
of restriction mapping.

Best wishes,
-- 
Chris Boyd                       | from, | MRC Human Genetics Unit
chrisb at hgu.mrc.ac.uk             |  not  |  Western General Hospital
http://www.hgu.mrc.ac.uk/~chrisb |   for |   Edinburgh EH4 2XU, SCOTLAND



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