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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
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