Uncomplete plasmid after transformation - (Jan/31/2008 )
In first, I would like to say that this forum is a very good idea ;-)
My problem is that I get trouble with my transformation. I would like to transform a recombinant plasmid of 12kb which expressed a toxic viral polyprotein for the bacteria (I used Dh5Alph). Bacteria must grow less than 16hours in this case. I transformed with eletroporation system. I have already done it with the same plasmid (mutants with 1/2/3 mutations in specific regions) and it worked well. Now I need to do the same with the same plasmid but without a viral gene (plasmid PGEM+viral genome without one viral gene), the problem is that after transformation, I obtained a lot of uncomplete plasmid, with lower size (I compared the 3 forms with a positive control),and when I digest them,I get a crazy profile of digestion. It's like the bacteria modifies this plasmid because it's too toxic. I obtain diferent phenotype of bacteria,big one has a little truncated plasmid, little one has a bigger truncated plasmid but more little that I hoped.I used a Ampicillin concentration of 100 µg/mL on Petri plate.
If you have any idea...Maybe I have to change bacteria strain...
Weird DNA profiles can occur if there has been some recombination events. Some sequences promote it and some can avoid it. Removal of the viral gene in your plasmid could have promoted this event. try to transform in bacteria which prevents recombination like SURE cells (Stratagene). Also ensure the antibiotic in the agar plate is new and not degraded.