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Sv: Help?

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Wed Jul 28 06:17:31 EST 1999


Hi Gregers, Hi David,
Cloning into the kan-resistant EGFP-vectors is quite robust. I found it is not
a "difficult" vector, so I´d assume you don´t have to screen a large number of
clones. In my last clonings into EGFP-N1, I picked 24 colonies for minipreps,
and there were usually more than 10 that were ok. As always, directional
cloning is useful. I didn´t dephosphorylate, and I found that a gel-purified
vector gives very low background anyway.
Some tips for cloning into this vector:
1. After transformation, do not allow colonies to grow for more than 12h.
Usually, kan-resistant vectors produce a background of slowly growing colonies
that DO NOT have any plasmid. So, if after 12h there are no colonies, don´t
wait longer but discard plates. Making minipreps from these slowly growing
colonies is wasted time, they never contain what you want.
2. If possible, you can select for insert-containing constructs by a
restriction digest after ligation, but prior to transformation. Choose an
enzyme that does a.) not cleave in your insert but b.) cleaves a site between
the two sites you use for cloning. Any vector that has been religated will
therefore be linearized and will not give rise to a colony.

Hope this helps,
Frank


Gregers wrote:

> Hi David,
> Cloning in Kanamycin plasmids gives me a hard time. What is your cloning
> background (ligation w/o 1,6 kb insert)? If this is close to the number with
> insert then try dephosphorylating the linarized plasmid with a phosphatase
> e.g. the
> heatinactivatable Shrimp Alkaline Phosphatase (SAP from Amersham Pharmacia
> Biotech). This
> should increase the ratio of "correct" clones.
>
> Best
> Gregers
>
> "DAVIDS, L, LESTER, MR" <lester at uctgsh1.uct.ac.za> skrev i en
> nyhedsmeddelelse:E117ezV-0001Mb-00 at mail2.uct.ac.za...
> > Hi All,
> >
> > I am currently using pEGFP-N1 as my vector of choice. I plan to
> > sticky-end ligate a 1.6kb gene into the MCS of this vector. I'm
> > wanting to know whether anyone knows if this vector has some other
> > selection marker besides the antibiotic resistance genes as I'm
> > getting 300+ colonies and it would take me well into the next
> > millenium to scrren them all for a positive one. If there is no other
> > marker, what about the idea of ligating in lacZ next to my gene into
> > the GFP's MCS?
> >
> > Please let me know if anyone can help!
> >
> > Thanx,
> > ********************************************
> > Lester M. Davids,M.Sc(UCT)
> > Lennox Eales Porphyria Laboratories
> > MRC/UCT Liver Research Centre
> > K-FLoor, Old Groote Schuur Hospital
> > UCT Medical School
> > Observatory, 7925
> > South Africa
> > Tel. 27-21-406 6519
> > Fax. 27-21-447 9670
> > :-) "Chance befalls the prepared mind" :-)
> > ********************************************
> > ---




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