plasmid contamination - (Mar/07/2006 )

dear people,

i have a contaminated plasmid (or so i thought), but i'm not sure why it happens to be one. i keep the plasmid (which i used as vector in my cloning work) in glycerol stock and in plasmid form and keep it in the -20 fridge.

i realized it have been spoilt when i transformed it as one of my positive control together with my ligation mixture. the plasmid have been fully plated on it's selective antibiotic (fresh 50ug/ml LB-kanamycin), but also appear about 20 colonies on other plate (freshly 100ug/ml LB-ampicillin).

another problem is that i have been using the plasmid as vector, and the preparation of the vector (isolated, digested, and purified) was done using the same source of plasmid, in my new cloning work. for your info, i haven't use/touch that plasmid about 11 months ago. the subjected ligation plate appear to be positive on wrong selective plates(ampicillin) but none appear on the kanamycin plate.. it that because the vector itself was spoilt?or contaminated?

is there any way to decontaminate the plasmid?please help me.

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hi
you use 2-3 enzymes that do not cut in your plasmid. Purify your plasmid on gel and electropore it. Do minipreps and you'll certainly be able to find your plasmid.

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thanks fred.

but instead of using electroporation, can i use heat-shock transformation?

one more thing, do u mean i have to double digest or just digest with diff. enzymes in diff. reaction?

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