User:Linh N Le/Notebook/2009/06/23

To Do

• Koch/Atlas group meeting at 3
• Try out new buffer we made yesterday?
• Try Kinesin?
• Clean up lab

12-6

BRB80

• Since there are so many ways to make this buffer, Koch wanted me to try to find different famous people's recipes for the buffer
  • People to look for, Block and Vale
• What we use for Cytoskeleton is called PEM and while PEM and BRB80 can be the same, they do not have to be
  • I did searches on both PEM and BRB80 and the following recipes are also reported by what they are called
• Special Note: All PDF's linked here are from the personal website of the authors and are free access

Recipes

• Hancock (Used by us as well)
  • 80 mM PIPES pH 6.85 (we use ph6.9)
  • 1 mM MgCl2
  • 1 mM EGTA
• T.Salmon Labs
  • PM Buffer
    • 100 mM PIPES, pH 6.9
    • 2 mM EGTA
    • 1 mM Mg2SO4
• Goldman Lab (Northwestern University)
  • 100 mM Pipes,pH 6.9
  • 1 mM MgCl2
  • 1 mM EGTA
• Cold Spring Harbor Labs
  • PEM Buffer
    • 100 mM PIPES (pH 6.95)
    • 2 mM EGTA
    • 1 mM MgSO4
• Cytoskeleton
  • PEM (General Tubulin Buffer)
    • 80 mM Na-PIPES pH 6.9
    • 1 mM MgCl2
    • 1 mM EGTA
• Mitchison Lab (Harvard)
  • Brinkley Buffer 1980 (BRB80)
    • 80mM PIPES pH 6.8
    • 1mM MgCl2
    • 1mM EGTA
  • "Cytoskeleton Buffer"
    • 10mM MES pH 6.1
    • 138mM KCl
    • 3mM MgCl
    • 2mM EGTA
  • Notes: BRB80 is good for microtubules and 'cytoskeleton buffer' is good for both actin filaments and microtubules
• Sigma Aldrich
  • PEM Buffer:
    • 0.1M PIPES (Product No. P8203)
    • 5 mM EGTA (Product No. E4378)
    • 2mM MgCl2 · 6H2O (Product No. M0250)
    • Bring to pH 6.8, using NaOH solution
• Bayer College of Medicine
  • PEM Buffer:
    • 80mMPotassium PIPES, pH 6.8
    • 5 mM EGTA, pH 7.0
    • 2 mM MgCl2 ( final Concentration: 2 mM)
• Yu-li Wang from Carnegie Mellon This is a pdf of how they do Rhodamine Tubulin
  • PEM buffer:
    • 0.1 M PIPES
    • 1.0 mM EGTA
    • 0.5 mM MgCl2
    • pH 6.9
• Tedeschi of Italy
  • PEM buffer
    • 85 mM Pipes, pH 6.94
    • 10 mM EGTA
    • 1 mM MgCl2
• Steve Gross of U Minnesota with Steve Block
  • 80 mM Pipes
  • 1 mM EGTA
  • 4 mM MgCl2
• Stefan Diez (Germany)
  • BRB80 buffer
    • 80 mM potassium Pipes
    • 1 mM EGTA
    • 1 mM MgCl2
    • pH 6.9
• Mathew Lang, Steve Block first author
• Also used in Rosenfield, Block Paper
• Also used in Guydosh Block Paper with 10μM of Taxol
  • Un-named buffer
    • 80 mM Pipes (pH6.9)
    • 50 mM potassium acetate
    • 4 mM MgCl2
    • 2 mM DTT
    • 1 mM EGTA
    • 7 mMTaxol
  • Andy mentions that he thinks these buffers are what Block used to get kinesin to stick to beads. It not be what we want, but if the whole "soup" (Beads + kinesin + buffer) is added to the microtubes mix and it doesnt mess things up, this stuff should be fine.
• Valentine, Fordyce, Block
  • polymerization buffer for brain tubulin
    • 62 mM PIPES at pH 6.9
    • 0.8 mM EGTA
    • 3.7 mM MgCl2
    • 0.9 mM GTP
    • 10% v/v DMSO
  • stablilzation buffer
    • 80 mM PIPES at pH 6.9
    • 1.7 mM EGTA
    • 5.5 mM MgCl2
    • 1.2 mM GTP
    • 8.2 mM sodium azide
    • 20 μM taxol
    • 10% v/v DMSO)
  • Assay buffer based on PEM80
    • 80 mM PIPES at pH 6.9
    • 1 mM EGTA
    • 4 mM MgCl2