User:Linh N Le/Notebook/2009/06/22

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To Do

  • Andy is back! He wants to try the kinesin again, maybe his expertise will allow us to succeed
  • Make BRB80



  • Stumbled upon a site that details how they get tubulin from cow's brains


  • Since Andy and Koch are the only people in the lab that know how to make a buffer (I actually dont know if Ant can), we waited til Andy came back to try to make BRB80
    • Not to mention the fact that we are also running out of the stuff we got from Cytoskeleton (new shipment will be in this week)
  • Andy is looking over our new fancy pH meter
    • He is starting the calibration process
  • BRB stands for Britton-Robinson BufferSJK 01:52, 23 June 2009 (EDT)
    01:52, 23 June 2009 (EDT)
    I can't tell if you're kidding! Check out this friendfeed thread: what is BRB80?
    Linh N Le 19:31, 24 June 2009 (EDT)I googled "BRB Buffer" and thats what I found. I now know thats now what we want
    • It is a "universal" buffer whose ph changes linearly with the amount of alkali that is added to it
    • It consists of:
      • .04M H3BO3 (Boric Acid)
      • .04M H3PO4 (Phosphoric Acid)
      • .04M CH3COOH (Acetic Acid)
  • The stuff we use is also known as PEM
    • 0.1 M PIPES (pH 6.95)
      • Piperazinediethanesulfonic acid C8H18N2O6S2
    • 2 mM EGTA
      • ethylene glycol tetraacetic acid C14H24N2O10
    • 1 mM MgSO4
      • Magnesium Sulfate
  • Also can make it as
    • 100 mM Pipes,pH 6.9
    • 1 mM MgCl2 (magnesium chloride)
    • 1 mM EGTA
SJK 01:58, 23 June 2009 (EDT)
01:58, 23 June 2009 (EDT)
I notice a difference in 2 mM versus 1 mM EGTA here. The MgSO4 I was aware of (and worried about in the past)-- I think perhaps Block lab uses MgSO4. It's important to iron this stuff out. When you have time, it would be great to look up the famous labs and see what their PEM / BRB80 recipes are. Andy and I can help you identify labs to look at. Some resources so far: * Cytoskeleton PEM recipe * The actual recipe I used at Sandia * Recipe on kinesin home page (almost the same as cytoskeleton, but slightly lower pH]

Ph Titrations

  • Recalling what little i remember from freshman chemistry, Andy has instructed me to create 40uL of a "normal" base of NaoH
    • Normal meaning 1M soln
  • So, we look up the molar mass of NaOH
    • MM 39.9971 g/mol
  • If I want a 1 mol soln, i need 39.9971 g in 1L
    • Means I need 3.9971g in a dL
    • Means I need .39971g in a ml
    • Means I need .039971g in a ul
      • Meaning I need 1.598g in a 40ul soln
  • Thats wrong, new train of thought
  • Lets try to make .4L of 1M soln
    • I need 15.99884 g for .4L of water (4 deca liters)
    • divide by 10, 1.599884g for a 40ml

Making the Buffer

  • We measured out the following
    • 1mM EGTA (0.076g)
    • 80mM PIPES (4.83g)
    • 1mM MgCl2 (.2mL)
  • For a 200mL stock of the Buffer (which Andy hates naming, but I will name KLB)
  • We titrated it with NaOH to raise the Ph to 6.89
    • Used about 26ml of NaOH to get there
  • Added an entry to the Kochlab inventory page, under the VWR fridge category (Steve Koch 02:07, 23 June 2009 (EDT): Thank you! Excellent notes, as always. I really appreciate it.)