Is it possible to transform competent cells with linear DNA? - (Aug/03/2007 )
Hi, I have done transformation of HB101 competent cells (promega) with plasmid DNA before. I am wondering if it is possible to transform with linearised DNA? The DNA was cut with two specific sites that I want to retain. Will the transformation alter these sites? Will the linear DNA become circularised after transformation? Any help would be appreciated!
I donot think you will get any colonies if the linearised DNA is transformed. The plasmid will not religate itself.
linear DNA is assumed as forign DNA and destroyed by the cell.
It cannot religate itself in the cell, and so can't replicate.
nope not really.
The cellular endonucleases will break up most of the linear DNA strand.
There is a small chance of homologous recombination, between sections of the plasmid which are similar to each other (especially if plasmid contains tandem array). This can save plasmid but scrambles its structure. Not much use.
There is the possibility of the plasmid recombining with the bacterial genome... again not useful.
Thanks for all the advice!
Plasmid linearized with Hind III is capable of being transformed with good efficiency by E. coli strain C600. I had to dephosphorylate the ends to prevent this from occurring.