Sometimes science isn't linear (and I'm not talking about exponential growth)

Sometimes science (my project) isn't linear, and I've been getting caught up in this recently with my posting.

I wanted to do a series of posts and videos on the process of transformation and the completion of my first NiR plasmid, but what I found was that sometimes things don't work out the way you want.

So, let me backtrack a bit.

As I mentioned before, it appears I have transformed bacteria colonies that have my PCR insert. Great! But I've been having trouble getting the insert to amplify out of the plasmid once again. I grew up several bacteria colonies that looked like they had my insert (white colonies on X-gal) and performed a plasmid prep that yielded very little plasmid DNA. I need a decent amount of this plasmid to allow me to digest (cut) out and obtain the insert.

Now, I'm trying to do yet another PCR reaction in a much larger volume (50µl rather than 10-20µl) using the plasmids I obtained from my lame plasmid prep. If this works, I'll have a lot of copies of the NiR terminator insert, which I can then slice off the ends with restriction enzymes. Then, the insert would be ready for the next step.

That is, if I can get this to work. :-\