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Transformation with a big plasmid - (Sep/13/2012 )

I'm trying to construct a plasmid to get a mutant by double recombination and insertion of an antibiotic cassette in the place of the gene i want to knock out.
In the process, I need to cloned the up and down regions of the gen of interest, and then insert the cassette in the middle.
I'm using the pGEX3x (4900bp), and i have just cloned the up (1000bp) and down (1000bp) regions. I cut the plasmid obtained, with HindIII (between the regions up and down) and i observed that it has the expected size. Then when I ligate it with the cassette ( Stp, 2000bp) and transform the JM109, I can't get bacterias with streptomycin resistence.
I wonder if the size of the plasmid (9000bp) could be hindering the transformation.


It may be a factor, but 9000 is not extraordinarily large. More likely your cells are less than ideally competent, or your strep resistance cassette has incorrectly cut ends. Have you confirmed that your strep cassette can be selected for in JM109?


Is your streptomycin resistance cassette correct expressed?
Do you have promoter, start codon, stop codon (...), all without interference of the 1000pb upstream and downstream?


Did you use X-gal and IPTG in your selection media. If you don't use IPTG your inserted gene(streptomycin resistance gene) wont be expressed because the plasmid contains tac promoter and also contains lacI which inhibits the promoter. So, check this step. On the other hand i would suggest you to select your transformed cell in double antibiotic(Ampicillin and streptomycin) as your plasmind contains Amp resistance.


The streptomicyn cassette contains its own promoter, start codon and stop codon.