Generating stable cell line with linear DNA - (Mar/05/2012 )
I'm planning on generating a stable cell line using linearized plasmid DNA. Is there a risk of having my DNA re-ligate? If so should I treat my DNA with alkaline phosphatase after digestion? Please let me know how I would do this as I am new to all of this.
Also, is there a difference between blunt and sticky end digestion?
There is a small risk of re-ligation, though you are more likely to get concatamerization, and the amount of re-ligated DNA without adding a ligase will be very very low, so I wouldn't worry about it.
It shouldn't matter whether you use blunt or sticky, but blunt is less likely to re-ligate.
Thanks bob1. Fingers crossed.