Yeah, as I said, probably the enzym didn't work well.
As for the 1ug we just used 500 ng in the first attempt. And you could hardly see the bands.... So my guess is that the two bands were supercoiled and circular plasmid, and the amount of cut plasmid was too small to be seen.
However, we are doing a midiprep to be able to try it again.
On Fri, Feb 1, 2013 at 3:07 PM, Dakota Hamill <dkotes@gmail.com> wrote:
Ah yeah I recall reading the post about the blunt ends and the fear of poor ligation efficiency. Did you give the ligation a try, then follow it with a transformation? 1ug seems like it'd be enough at 20-50ng per transformation, though I don't know the amount needed per ligation.
Still, all you need are a few colonies that pass the screen test and you can culture them up and mini prep out the plasmid
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