SHOTGUN SEQUENCING

Dr. C. Dolphin cdolphin at crc.ac.uk
Tue Dec 7 07:30:53 EST 1993


In article <1993Dec7.084144.4413 at crc.ac.uk> mjones at crc.ac.uk (Dr. M.D. Jones) writes:
>Hi netters
>
>One of the favoured ways of sequencing large DNA fragments is to shotgun clone 'sonicated'
>DNA into a M13 vector and sequence the clones, and compile the data by computer.
>
>In the past I have successfully used this strategy, but the most frustrating steps were getting
>enough recombinant clones to complete the sequence.
>
>This is due to the damage caused by sonicating the DNA, making most of it unclonable.
>
>Does anybody out there have a detailed fool-proof 'sonication' protocol ?
>
>Anybody have any advice/tips/etc on how to get the best out of this procedure ?
>
>On a similar note, does anybody use 'partial' restriction enzyme digestion to
>fragment their target DNA ?
>
>Fitzgerald et al, 1992 (Nucleic Acids Res., 20(14), 3753-3762) used the enzyme
>CviJI (recognition sequence RGCY) to create essentially random shotgun DNA for
>sequencing.
>
>Does anybody know if this enzyme is commercially available, particularly in the UK ?
>
>Many thanks
>
>
>Mick Jones
>
>Department of Virology, RPMS
>Du Cane Road, W12 0NN, UK
>(voice) 081-740-3328
>email: mjones at rpms.ac.uk
>



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