No wonder I am going gray

I don't have to kick myself. The one thing that I thought would fix my Merlin problem, didn't. I figured that I was heat shocking the bacteria too long and that they were all dying. It made perfect sense. Yesterday, I decreased the time I heat shocked the bacteria but when I came in this morning, there was no growth on any of the plates. This afternoon, I am going to try a different approach. Instead of using heat to open up the bacterial wall, I am going to use electricity. If this doesn't work, I am pretty much up against a wall.

Dad, you said that you didn't understand what I have been talking about. Let me attempt to explain it. What I am doing is essentially editing. I am taking a sentance from one page, cutting it out of its context, and pasting it into a new paragraph with a new context. Instead of words, however, I am doing this with DNA. I begin with a process called PCR that "photocopies" the stretch of DNA I am in which I am interested (in this case, a gene called Merlin). This strand of DNA is then pasted (called a ligation reaction) into another strand of DNA (a circular contruct called a plasmid. Plasmids contain regulatory elements that allow my gene to be expressed efficiently) that has been cut open (this process is called a restriction digest) at sites that are complimentary to the ends of my DNA strand of interest. Now I use nature to make more copies of my finished product. I do this by introducing my plasmid into bacteria (usually a strain of E. coli) in a process called transformation. Finally, I plate my transformed bacteria onto agar plates and incubate them over night. The problem I have been having is that I am not getting any colonies on my plates, not even in my positive controls. Hopefully this explaination has been helpful.