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Failed Transformation of Tuner (DE3) cells - (Oct/29/2009 )

I don't know if you are familiar with the Novagen kit.
I ligated a fusion PCR Product (1750 bp) into the pET-Blue Acceptor Vector then transformed it into Nova Blue.
This step had to be repeated around 5 or 6 teams before i got one positive clone.

Then i extracted the plasmid from my positive clone with the Fermentas mini prep kit
then i transformed 1ng of the plasmid into the Tuner (DE3) pLacI cells

I used a positive control along to compare

my positive control gave the expected number of colonies
while my plasmid didn't lead to any colonies. Nothing

The thing is i have added IPTG to the plates, and i was advised not to.
But the positive control did grow on IPTG.

and the thing is the control has the 1000 bp cellulase gene
whereas the new plasmid has the 1000 bp cellulase and a 750bp CBM = 1750bp long gene

given that the CBM has no catalytic activity, its only a scaffold, i doubt it is toxic!

So is there any other explanation why my plasmid of interest wasn't transformed into the cells?
although the protocol is very easy one

and i have been using it for the past year, and it has NEVER ever before not worked

HELP, ideas? advices? please
Thanks :)


I experienced that problem many times when trying to transform Tuner cells with a supercoiled DNA coming out of my Novablue positive clones !

Just repeat several times with different amount of the plasmid. The theory says that 1ng is more than enough but the experience says use more it might work better :)

Do you have a second antibiotic resistance in your Tuner celles like a Lac repressor plasmid ?