I have a lot of work to do with regard to organizing my thoughts for this project. It has been 1 year since I last thought about this, but it is time to restart it. I’ll explain the history of this project and where I’m going with it in future posts (this week), but for now I’m going to introduce what I need to do this week with some links for me to check out.
Here is an intro to Shotgun DNA Mapping. Go there for a crash course and some links that go further in depth than what I’m ready to divulge at this moment.
Step 1 of Shotgun DNA Mapping (SDM from here on out) is to create the unzipping construct. For that I’ll need some DNA.
There are 3 pieces of DNA required to have a completely unzip-ready object. They are:
- The anchor – this is a 1kb/4kb (depending on the situation, kb = kilobases) double stranded sequence that is created from the PCR reaction of a plasmid. In our old cases it was pRL574, but I experimented with another sequence that I named pALS. I may start with the pRL574 plasmid to get started. This piece contains a molecule that allows us to attach the DNA to a glass surface (the microscope slide).
- The adapter duplex – this is technically two single strands of DNA that are annealed together to create a weird double stranded piece. It is ~25bp long and there are tons of variations that I’ve experimented with. This piece contains a molecule that allows us to attach the DNA to a microsphere. It also hosts a space that allow us to essentially break the DNA so we can unzip it.
- The unzipping DNA – this is the DNA that gets unzipped in the experiment. It can be anything essentially, but for the purposes of SDM we use yeast genomic fragments, and in the very near term I’ll be using pBR322 (a commerically available plasmid) to test the reactions and to calibrate the tweezers.
As I start to figure out what needs to be done I’ll have separate posts explaining everything about each piece. In the mean time here are a bunch of links that will help organize my thoughts:
- pALS sequence and design
- pALS PCR primer designing
- another pALS primer (an update on the previous link)
- alternative adapter design
- unzipping ligation with some planning – I don’t really know what to call this link, but at the bottom there is some potentially useful information
- resuspending oligos – not useful for this experiment, but contains information that will help me look for other stuff
- DNA sequences – this should be the page I lean on the most
The order of things that need to get done:
- I need to check my inventory. I’m not going to use this stuff for the most immediate experiments, but it will be good to know what I have. I will need to use my supplies of pALS and pRL574 and pBR322, but the adapter sequences will need to be brand new.
- Check the DNA sequences.
- Order new sequences.
- Get into molecular biology – PCR, annealing, ligation, gels, digestions, etc. This is where it gets exciting.
I’ll start by posting pictures and descriptions of the things that I have.