User:Anthony Salvagno/Notebook/Research/2009/11/03/Redoing Unzipping: pBR322 Digestion and Extraction

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I probably won't get to this until this afternoon, because the annealing is occupying the PCR machine and going slowly. (Steve Koch 19:08, 3 November 2009 (EST): Maybe for next time, you can step the temperature a little more quickly.)

Setup

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Gel

I set up a 0.8% agarose gel. I will run it at 140V for a length of time that I determine to be adequate for band separation (maybe 20 min). Gel image below. I will then gel extract the large band and clean it.

Apparently there wasn't 30ul in the tube after addition of 5ul of loading dye. I'm gonna estimate around 25ul total. That is lame, but whatever. I hopefully have over 15ug of pBR322 digested. If that is my assumption, then I can fit it all into one column because I will not have more than 10ug of half a plasmid.


I let the gel run for longer than I expected to, which finally gave me sufficient spacing. As you can see the stain was starting to get pulled away from the DNA, but it still sufficiently labeled the pBR322. Lane Analysis:

  1. 1x ladder
  2. 2x ladder
  3. 3x ladder
  4. digested pBR322

You can also see, although not well that it was mostly a complete digestion, but not completely. I don't think that is too bad though. That red shit on the right is an artifact of the filter. Time for extraction.

SJK 19:07, 3 November 2009 (EST)
19:07, 3 November 2009 (EST)this looks like really good digestion, I can't even see the initial plasmid band, but I wouldn't worry about it.
19:07, 3 November 2009 (EST)
this looks like really good digestion, I can't even see the initial plasmid band, but I wouldn't worry about it.

Results

pBR322 - 53.1 ng/ul --> 32nM

Yay for me!! Although I got a really shitty yield. If I had 15ug of DNA to start and then after digestion I would get ~9.3ug, this yields a 40% yield. That's lame but at least I have something to do tomorrow.

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