Hi, Experts,

need you help!

I try to subclone two genes(PCR Product) into PET-28a (+) using HInd III and BamHi digestion sites.

Untill now everything is fine, I got the correct ligation products which are verified by double digestion and PCR for T7. About 10 clones/gene have been verified.


I got a lot of plasmid by transforming the plasmid into DH5a.
Now I have my problem, I transformed the plasmid into BL21 (AI) and got colonies on the plate while nothing in the medium, looks like the expressed bacterial can grow on plate but difficult in the medium.

Anyone has any suggestion or any analysis?

Thank you so much!

Kelly

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Hi Kelly
This happened in our lab, too. I recommend to prepare fresh plates. It seems, that the antibiotic in your plate is degraded or too old. (Than bacterias loose their plasmid).
This worked in our hands. Wish you good luck
Cheers
mue

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Thank you for your reply!
But I guess maybe that is not the reason.

Actually I use the same tube antibiotics to prepare the plate and the medium. I just poured the plate on the same day I do the transformation.

Those plasmid grew good both in the plate and in the medium if they are transformed into DH5a cells.

kelly

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