competent cell contamination? - (Dec/31/2007 )

Resently, I've made competent cell for at least three times.

But I always got the same outcome...my competent cell(XL-1 blue) without else plasmid is resistant to kanamycin.

Before making competent cell, I would struck XL-1 blue stock (is not resistant to Kan originally) on LB plate with tetracycline.

Then picked out one colony from the plate and innoculate about 5ml sterile LB overnight . Next day transfered it to 500ml LB w/o any antibiotics.

For testing my competent cell, I would plate them on LB agar plate with amplicillin and kanamycin.

There should not be any colonies growing from both of these plates.

Amplicillin plate do not show any colony, however, the kanamycin one grew so many colonies.

Unfortunatelly, I have to use kanamycin to select my ligation products....

I am still looking for the reason, my operating way? environment contamination?? or any else possibility??

Could anyone give me some advices??


Thanks and Happy New Year!

Are you 100% sure about the kanamycin stock concentration and that it's active?

Are you sure you don't have XL2-MRF' cells instead of XL1-Blue? Those are KanR (and TetR, and CmR). One wonders what Stratagene is thinking.

Yes, I am pretty sure that I use XL-1 blue 'cuz I take stock from commercial competent cell.

I tested the commercial competent cell by plating it on a kanamycin plate, and the plate didn't grow any colonies, thererfore, I am also sure that antibiotic is still active.