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Sv: Help?

Gregers gregers1XremoveX at hotmail.com
Tue Jul 27 02:14:44 EST 1999

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.


"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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