User:Anthony Salvagno/Notebook/Research/2010/05/03/Project Lambda is a Go

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I will be getting this project off the ground today now that I have all the necessary parts. I will start with a little organization and math and then setup the reaction.

Contents

Prep

View/Edit Spreadsheet

Experiment

View/Edit Spreadsheet

Notes

Some things to note:

  • Koch found a useful website for dealing with rate constants here.
    • I based the amount of dNTPs to use on that page
    • I noticed (after the fact) that there was no value in the column for DNA and I took that as using the next available value (above it) which is 5nM. This is precisely the amount I used and so the Klenow Fragment will probably proceed a little slower. I will have to give the reaction more time next time.
  • I didn't think of this, but I should preheat the lambda DNA to detach possible ends from each other since they are complementary.
    • Steve Koch: Oh yes, I was told that's important back in the day. Hopefully it was at least a bit warm?

Reaction Analysis

There is a good chance I don't get to this today because of various circumstances, but here is what I would/am going to do:

  1. Purify via Phenol:Chloroform Extraction and EtOH precipitation
    • I'm not sure the P:C is needed, but might as well do it.
  2. Set up a gel
  3. Put Neutravidin in solution with lambda DNA and incubate for 10 min
  4. Run gel of protein/DNA and just DNA and compare
  5. Either do over or give to Collaborators

I got to the point of precipitating the DNA in EtOH and that I can do indefinitely. Since I won't have time to finish I will just leave it there and come back to it next week. See you soon lab.

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