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Transformation issue


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#1 rjthomp2

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Posted 09 June 2011 - 07:00 AM

Hello all,

We are trying to trasform e coli for plasmid amplification to use to transfect mammalian cells in culture. The plasmid encodes carbenicillin resistance, in addition to our gene of interest. Our gene of interest has been subjected to site directed mutagenesis to change one base in order to get a different amino acid encoded by that codon in the mammalian system. We have new competent e coli, new pfu, and the plasmid does amplify in PCR, but when we go to transform the competent cells, we are getting no growth on carbenicillin containing medium, but in our positive control plasmid (the wild type gene) transforms and grows on the medium just fine. Therefore, it seems that there must be something going wrong with the transformation of the mutant plasmid... but how could that be? The protein is not getting produced in e. coli because the promotor for the gene of interest is mammalian so how could mutant DNA affect plasmid amplification? It could be possible that there is a problem with the expression of the antibiotic resistance, but why would that happen ONLY for the mutant plasmid but not the other plasmids?? Help!!

#2 GNANA

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Posted 09 June 2011 - 12:48 PM

have you completely sequenced the mutated plasmid??? one possibility is your induced mutation or even PCR (if your tag is low fidelity) could have altered the ORF of the antibiotic resistance...check for that..

Gnana..
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....




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