Sticky ends and blunt ends in DNA cloning?

[scilearner]

 

1. Ok in the plasmid we used the ECOR1 to create sticky ends. Now my question is when we add DNA ligase, why don't these ends stick back again. How are we able to add new DNA piece in between. Basically what I'm saying is when ECOR 1 creates sticky ends in plamids, why do they separate the plamid, don't the sticky ends stick back together.

 

2. Also why can't DNA ligase bind the blunt ends, can't it fix the phosphodiester bond there.

 

Thanks

[CharonY]

There is a chance that it re-ligates. In fact, more will be re-ligated vectors rather than vectors with insert. This is what the selection step is for (e.g. blue-white). Religation rate can be reduced by dephosphorylation, though.

Blunt ends also ligate, albeit with lower efficiency than sticky ends.

Edited January 8, 2011 by CharonY

[Genecks]

I can't remember, but I think there is a mathematical name for the percentage of plasmids that take in the gene.

"Transformation efficiency" comes to mind, but I don't think that is it.

Edited January 9, 2011 by Genecks