directional cloning problem!
Michael L. Sullivan
mlsulliv at facstaff.wisc.edu
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 .
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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