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M15 transformation problem? - (Sep/18/2005 )


I am trying to transform M15 with my construct , which contains my PCR product in PQE30 vector.I conformed presence of my insert by RE. But facing problem in M15 tansformation step.I used PQE30 only as control to check whether my insert is toxic to the bacteria.I do not find any colony there. M15 competent cells grow well in absence of any antibiotic or in presence of Kanamycin 25 microgm/ml concentration.
Now i am confident , my insert is not toxic atleast. Either M15 failed to takeup plasmid due to its lost competent factor or the antibiotic which I used in plate were in higher side. Can anyone suggest me how to deal with M15 expression host? Please let me know abt Incubation time , which media do u use after heat shock ............

Thanks in advance.

Saikat Chakraborty

Mie University , Japan


Use electroporation. It is much less sensitive to difference in competency between strains.


DNA sequencing

-Daniel Tillett-