User:Anthony Salvagno/Notebook/Research/2009/09/15/Digestion of pBR322 aka Fuck Yea pBR322 arrived today aka the Ballad of Earl and his battles with a 4kb monster

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Procedure

  1. Digest pBR322 with EarI
  2. Run gel of digestion
  3. Extract digested product
    • There are 3 cut sites in pBR322 resulting in 3 fragments. I want to extract the longest piece.
  4. Check in nanodrop

Setup

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I am digesting 10ug of DNA (1ug/ul) with 2ul of enzyme. Technically I can get away with 1ul because there are 20u/ul, but I don't know about the concentration (10ul DNA in 20ul reaction) so I added an extra ul for good measure. Reaction time is 60'. I also am not using BSA, and the reason is I don't think you need to. NEB redid their whole buffer system so every enzyme uses buffer 4 and I think that's all you need. It's in the instructions. I should have done a test digestion but I don't have time for that now.

Gel

pre-gel analysis: I found online that pBR322 only has 2 cut sites with EarI. I find this strange and disturbing. If this is the case how will I know which piece to extract?

Capture 00011.JPG

This is an image of the gel while the gel is still running. I thought that was pretty sweet. Only I have the power to do this in the entire world! Maybe. Anyways, looks like there is only 2 sites. What to do?

Extraction Data

I am extracting both fragments and will decide the proper course of action later. Nanodrop Readings:

  • Short Strand: 53.6ng/ul-->1567ng
  • Long Strand: 73.6ng/ul-->2060ng

This is sad because I started with 10ug of plasmid. Maybe the nanodrop is off? There is a large peak at 230, and I don't know what that means.

Steve Koch 15:03, 16 September 2009 (EDT): I don't know about the peak (maybe buffer), but this sounds about right. It's really really tough to get 80% gel extraction efficiency, and you may as well shoot for 50%. In your case, you got about 35% efficiency it seems. What you can do in the future is use a lot more plasmid. Knowing that 10 micrograms can stick to the column, you could have started with 20 micrograms, knowing that roughly half is going into each fragment.