User:Anthony Salvagno/Notebook/Research/2009/02/09/Plasmid Vectors and Cloning

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Kelly and I began discussing what vector we should use for the cloning phase of the research. He even had some pretty good ideas as far as tethering is concerned. I also learned some important things about what we hope to achieve and I will discuss all that here.

The Use of Plasmids

Restriction enzymes are very useful. We will use an enzyme that has one binding site in the plasmid we choose, cut the plasmid, and then cut our genomic DNA with the same enzyme. This will allow us to insert the fragment into our plasmid for cloning and tether creation. The enzyme has to create palindromic sites for this to work. I used Kelly's representation for the plasmid and linear DNA. I made up a sharp toothed monster for the RE and a cool chromosomal X for the chromosome (cause I have 1 X chromosome).

I'm running low on time cause I gotta get to my basketball game for warmups and stuff, but let me try and jot down some quick notes.

We want to use a plasmid that has one restriction site in it (for a given RE). We will use that enzyme to cut the plasmid and then insert fragmented genomic DNA into that site (the DNA also will be cut using the same enzyme). I'm unsure if the site has to be palindromic or not. I feel like it does for this part, but for later steps we want non-palindromic. Anyways, after this we will use CIP (calf intestinal phospatase) to remove the phosphate groups from the plasmid so that it cannot recircularize. This will allow our phosphated fragments to insert into the plasmid and we can do a couple things from there (clone and tether).

Kelly had some interesting ideas for the tethering. He wants to use a completely different enzyme to cut (again one site in the plasmid) just before and nearly adjacent to the fragment. This way we will have a section of dsDNA that is the same no matter what fragment we choose from. I called it a calibration fragment. I figured Larry could incorporate this sequence of DNA (we would supply it) into the sim and this could help the matching algorithm a little bit.

The beginning of the data is always crazy and I figure with this calibration DNA, the actual important DNA could be matched better since it is farther from the noisy simulated unzipping DNA. I will need to point this out in images if this is unclear.(Steve Koch 01:04, 10 February 2009 (EST): I think that's probably a sweet idea. This would be especially useful once Larry implements the stretching and sliding.)

Anyways, the second site would be nonpalindromic so that we could attach the tether construct from only one side. ALSO... since we would make this second cut with a different enzyme, there would be some cutting in the fragment that we need. This may/may not be a problem. In initial tests (unless the shotgun fragment is too short), this could be good because we would now have a more random fragment of DNA. Stay tuned... (Steve Koch 01:06, 10 February 2009 (EST): Great diagram and great page. The only extra thought I have is whether the vector to use could be optimized so that your methods would work with any interesting genomic libraries. BAC libraries or other things that I don't know enough about. Probably no matter what your methods will easily be adaptable to existing libraries including the things I just mentioned and cDNA, etc.)