Transformation with a big plasmid
Posted 13 September 2012 - 10:50 AM
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.
Posted 13 September 2012 - 12:28 PM
Posted 13 September 2012 - 01:21 PM
Do you have promoter, start codon, stop codon (...), all without interference of the 1000pb upstream and downstream?
Posted 16 September 2012 - 07:15 PM
Posted 19 September 2012 - 12:19 PM