Fragility of plasmids: size limit for shaking/vortexing?
Frank O. Fackelmayer
fof1 at chclu.chemie.uni-konstanz.de
Fri Feb 7 08:29:40 EST 1997
In article <32F88C97.1431 at sheffield.ac.uk>, K.Mulcahy at sheffield.ac.uk wrote:
> following restriction digestions with heat-resistant restriction enzymes
> and CIAP, I have to phenol:chloroform extract the samples in order to
> remove the enzymes. Some of my fragments are only 1.5 kb and seem to
> tolerate quite vigorous shaking by hand (necessary to for efficient
> extraction by phenol:chloroform). However, one of my plasmids is 10.5
> kb. Does anyone know if plasmids (pre-linearised) which are this large
> can be shaken vigorously without causing cleavage of the linear DNA
> fragment? If not, then what is the best way to extract them with
> Many thanks,
> Kevin Mulcahy.
Don´t worry about damaging your DNA if it is less than 50kb. Even lambda
DNA (or fragments of genomic DNA with similar size) does not break easily.
Anyway, there is no need to shake your samples vigorously during
phenol:chloroform extractions. Gentle mixing for several minutes is
sufficient, and, used in genomic DNA preparations, yields fragments over
100kB in length.
Hope this helps
PS: Don´t use kits based on adsorption of DNA to some solid matrix (glass
milk etc.). They WILL break your DNA when it´s over 10kb.
Dr. Frank O. Fackelmayer
Division of Biology
University of Konstanz
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