directional cloning problem!

Michael L. Sullivan mlsulliv at
Fri Aug 23 08:06:34 EST 2002

This may sound obvious (but plenty of people have made a similar 
mistake), but are you plating your transformation on kan and not amp 
selective media?  Don't forget that the pET28 series uses kanamycin 
as it's selectible marker!


>recently i performed directional cloning but can't make it. so i 
>want to know where the problem is. at first i made my pcr product 
>cut by NdeI and XhoI,then the plasmid i used is pET28b cut by the 
>same two enzyme. secondly the mixture of pcr product and the plasmid 
>after enzyme cut ligated with the T4 ligase.At last i can't get the 
>transformant on the plate.
>   so where is perhaps the main problem during the procedure? please 
>kindly offer your suggestion! thanks .
>      Carol


Michael L. Sullivan, 
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

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