User:Anthony Salvagno/Notebook/Research/2010/01/13/Super Secret Ultra Project
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.
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.
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:
- Forward - ATCCGGTAACTATCGTCTTGAGTC
- Reverse - CCTACATACCTCGCTCTGCTAATC
- 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: