User:Anthony Salvagno/Notebook/Research/2009/05/01/pAS lib

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Creation of Plasmid Library

Yesterday I created a vector/plasmid library from the plates that I made a long time ago (Steve Koch 13:08, 19 July 2009 (EDT):I'm guessing you used the double-digested version of the plate (EcoRI/XhoI)?). This is how I did this:

  1. Get some weird metal scraper thing (it is like a handle with a wire attached to it).
    • Be very sterile
  2. Using the scraper, scrape all the colonies from a plate. If there are a few you don't want, like blue colonies when using pBS, then try and avoid them. On some plates with high yield avoiding the unwanted colonies is tough so you can just get everything and don't worry about it.
  3. Inoculate in 2+mL of LB+amp.
    • At this point I used 400uL to make a frozen stock. Tube 59 in -80C freezer.
  4. Place in 37C shaker.

The point of this is to have tetherable random fragments from the whole rest of the genome instead of the 5 fragments that I made huge batches of. We cannot sequence these, obviously, but we can see if we can find a match using the tweezers. This could be a fun little exercise and another proof of principle.

Digestion

Since we have two anchor constructs, we need to do two digests to be able to ligate to the construct.

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