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NEED PROTOCOL OF MINIPREP - URGENT

Sergio sergioal at solea.quim.ucm.es
Wed Jan 19 16:57:30 EST 2000


Maybe the insert is very short and you don't see it in the gel. Try using a
different agarose percentage.
An expensive way to check a very small insert could be sequencing one of those
plasmid using any primer desingned for the vector.
If the insert you expect is large enough, you can check the supercoiled plasmid
inestead of the digested one, using a supercoiled DNA ladder as molecular weight
marker.
Another method would be the DNA dot (or colony) blot using a probe for the
insert, but it's a little long and depending on what you are doing (gene
library, subcloning, PCR cloning) could be innecesary.

Do you see, at least, the vector band in the gels??. In that case you don't need
a new DNA isolation protocol.



navita astuti wrote:

> Dear all,
> I'm Navita from Bandung Institute of Technology,
> Indonesia. I need some information about plasmid DNA
> isolation without any procedure of digestion with
> restriction enzyme.
> My experience is, when I pick 15 white colonies and do
> miniprep on them, but unfortunately when I digest them
> with restriction enzyme, they do not give any DNA
> insert.
> 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
>
> Thanks for your concern
>
> Regards,
> Navita
> __________________________________________________
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