User:Anthony Salvagno/Notebook/Research/2010/01/13/Super Secret Ultra Project

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Normally I'm all open science, but recently I got put on a miniproject (as recently as yesterday) and out of respect for my collaborators I will not reveal anything about their project. However, I am still open and will discuss everything that I am doing.

The purpose of this project is to make a molecular ruler. Without having the ruler be floppy (by making it too long) it will have to be shorter than DNA's persistence length. This is why we are aiming for ~30nm by making a 100bp oligo. There are a few ways to do this and Koch and I came up with a few yesterday:

  • PCR - this is the easiest because you just have to make 2 primers and let the process run its course.
  • PCR, digest, ligation - This is similar to the unzipping construct method. I will PCR a fragment that has a RE cut site. Then I will digest the fragment. Then I will ligate an anneal set of oligos to make the fragment the proper length.
  • Others

We are going to work on the first option for the time being, but if PCR doesn't work right then I will try option 2. Eventually something will work.

Designing Primers

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

I will be looking at notes from here.

I will use pBR322 as a starting point. I am looking to get a restriction site within the PCR fragment to give the users (mostly me) the option of cutting the fragment and making it smaller or longer if need be. I used the same Primer3 program and so far I got these results:

  • Primers:
  • Hybridization Tm = -16.5C - that is a weird number but Figure 1 will demonstrate that it is a low melting temp.
  • Self Hybridization:
    • Forward Tm = 10.3C (figure 2)
    • Reverse Tm = -1937C (figure 3)
  • Complimentary to plasmid (see figures 4 for forward and 5 for reverse): Tm = 69C for both primers with perfectly hybridizing. The pictures don't demonstrate this for some reason but the bases are complimentary.
  • Purely by luck there is a cut site for AlwNI. I've never used that before but it exists at position 2884. See figure below:

Total length of fragment is 99bp