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C Coward cc122 at mole.bio.cam.ac.uk
Mon Jan 24 12:53:11 EST 2000

arumanis at yahoo.com (navita astuti) writes:

<snicker snack>

>My teacher suggest me to find any procedure of
>detecting DNA insert without digestion with
>restriction enzyme. Is there any protocol for this
>purpose?? Please send me e-mail to:
>navita15 at bi.itb.ac.id

Very quick (and dirty method) - is fine if you're insert is big enough to
resolve from the plasmid without insert on an agarose gel:

1.Grow transformants overnight as normal
2.Pick colony and streak a single line across a fresh (selective) plate
(I used to do 10-15 lines on a plate). Grow overnight
3.Scrape the whole line up, suspend in 40 ul STE (NaCl plus TE, can't
remember the concentrations, but it probably doesn't make too much of a
difference. Recipe should be in mol. bio. methods books anyway)
4.Add 40 ul phenol:chloroform, vortex 30s, microfuge 5 min
5.Load supernatant on agarose gel (use about 10ul or thereabouts). Using
loading dye of course. I routinely prepared lots of samples by adding the
supernatant to loading dye in 396(?) well plates to save using loads of
eppendorf tubes.

For me this worked for high-copy plasmids in E. coli (I was using pUC).
You get a lot of pluming out of the agrose gel well due to residual phenol
but DNA was still visible. I screened many clones during deletion series
construction by this method and could reasonably readily resolve 500bp.


Dr. Chris Coward                       | Tel: +44 1223 333545
University of Cambridge                | Fax: +44 1223 766145
Department of Pathology                | E-mail: cc122 at mole.bio.cam.ac.uk
Tennis Court Road, Cambridge.          |        
CB2 1QP                                |
England                                |

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