Nov 09, 2022

Public workspaceBuffer Recipes V.1

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  • 1University of Pennsylvania
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Document CitationClark Fritsch 2022. Buffer Recipes. protocols.io https://protocols.io/view/buffer-recipes-bthknj4w
License: This is an open access document distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: March 19, 2021
Last Modified: November 09, 2022
Document Integer ID: 48396
Abstract
Recipes for a variety of buffers that I have used for my work.
Buffer Recipes -
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500 mM HEPES-KOH (pH 7.2)
Formula Weight = 238.3 grams / mole
Recipe: Add 119.15 grams of HEPES powder to 500 mL of milliQ water. Then titrate to a pH of 7.2 with KOH
and top off to a final volume of 1 liter with milliQ water.
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500 mM Tris-HCl (pH 7.6)
Formula Weight = 121.14 grams / mole
Recipe: Add 30.29 grams of Tris-Base to 300 mL of milliQ water. Then titrate to a pH of 7.6 with HCl and top
off to a final volume of 500 mL with milliQ water.
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500 mM MgCl2
Formula Weight = 203.31 grams / mole
Recipe: Add 25.4 grams to 250 mL of milliQ water.
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1 M Tris (untitrated)
Formula Weight = 121.14 grams / mole
Recipe: Add 30.28 grams to 250 mL of milliQ water.
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2.5 M KCl
Formula Weight = 74.5513 grams / mole
Recipe: Add 186.4 grams to 1 liter of milliQ water.
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Buffer S-300 (High Salt Buffer for Cation Exchange Column - eEF2 purification)
Components:
1. 20 mM HEPES-KOH, pH 7.2
- 40 mL of 500 mM HEPES-KOH (pH 7.2)
2. 10% glycerol
- 100 mL of 100% Ultra-Pure Glycerol
3. 5 mM MgCl2
- 10 mL of 500 mM MgCl2
4. 300 mM KCl
- 120 mL of 2.5 M KCl
5. 730 mL of milliQ water
* Add PMSF and DTT to a final concentration of 1 mM right before using Buffer S-300.
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Buffer S (Initial No Salt Sample Buffer for Cation Exchange Column - eEF2 purification)
Components:
1. 20 mM HEPES-KOH, pH 7.2
- 120 mL of 500 mM HEPES-KOH, pH 7.2
2. 10% glycerol
- 300 mL of 100% glycerol
3. 5 mM MgCl2
- 30 mL of 500 MgCl2
4. Top off to a final volume of 3 L with milliQ water.
* Add PSMF to a final concentration of 0.1 mM and DTT to a final concentration of 1 mM right before use.
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Buffer S-20 (Low Salt Buffer for Cation Exchange Column - eEF2 purification)
Components:
1. 20 mM HEPES-KOH, pH 7.2
- 40 mL of 500 mM HEPES-KOH, pH 7.2
2. 10% glycerol
- 100 mL of 100% glycerol
3. 5 mM MgCl2
- 10 mL of 500 mM MgCl2
4. 20 mM KCl
- 8 mL of 2.5 M KCl
5. Top off to a final volume of 1 L with milliQ water.
*Add DTT to a final concentration of 1 mM right before using the buffer.
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Buffer Q-40
Components:
1. 20 mM Tris-HCl, pH 7.6
- 40 mL of 500 mM Tris-HCl, pH 7.6
2. 10% glycerol
- 100 mL of 100% glycerol
3. 5 mM MgCl2
- 10 mL of 500 mL MgCl2
4. 40 mM KCl
- 16 mL of 2.5 M KCl
5. Top off to a final volume of 1 liter with milliQ water.
*Add DTT to a final concentration of 1 mM right before using the buffer.
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Buffer Q-500
Components:
1. 20 mM Tris-HCl, pH 7.6
- 40 mL of Tris-HCl, pH 7.6
2. 10% glycerol
- 100 mL of 100% glycerol
3. 5 mM MgCl2
- 10 mL of 500 mM MgCl2
4. 500 mM KCl
- 200 mL of 2.5 M KCl
5. Top off to a final volume of 1 liter with milliQ water.
*Add DTT to a final concentration of 1 mM right before using the buffer.
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eEF2 Storage Buffer
Components:
1. 20 mM HEPES-KOH, pH 7.2
- 10 mL of 1 M HEPES-KOH, pH 7.2
2. 1 mM MgCl2
- 1 mL of 500 mM MgCl2
3. 100 mM KCl
- 20 mL of 2.5 M KCl
3. Top off to a final volume of 500 mL with milliQ water.
*Add DTT to a final concentration of 1 mM right before using the buffer.
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eEF1A Storage Buffer
Components:
1. 20 mM Tris-HCl, pH 7.6
- 40 mL of 500 mM Tris-HCl, pH 7.6
2. 0.1 mM EDTA
- 200 uL of 0.5 M EDTA
3. 100 mM KCl
- 40 mL of 2.5 M KCl
4. 25% glycerol
- 250 mL of 100% glycerol
5. Top off to a final volume of 1 liter with milliQ water
*Add DTT to a final concentration of 1 mM right before using the buffer.
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Beads-Wash Solution
Components:
1. 1 mM HCl
- Add 41.32 uL of 12.1 M HCl to 500 mL of milliQ water.
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Wash Buffer 2
Components:
1. 0.1 M Na-acetate
- 4.1 grams of Na-acetate (F.W. = 82.03 g/mol)
2. 0.5 M NaCl
- 14.61 grams of NaCl (F.W. = 58.44 g/mol)
3. Fill up to 300 mL of milliQ water and then adjust pH to 5.0.
4. Top off to a final volume of 500 mL with milliQ water.
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Coupling Buffer
Components:
1. 0.2 M NaHCO3
- 8.4 grams of NaHCO3 (F.W. = 84.007 g/mol)
2. 0.5 M NaCl
- 14.61 grams of NaCl (F.W. = 58.44 g/mol)
3. Fill up to 300 mL of milliQ water and then adjust pH to 8.3.
4. Top off to a final volume of 500 mL with milliQ water.
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Quench (Q) Buffer
Components:
1. 0.1 M Tris-HCl (pH 8.5)
- 6.057 grams of Tris base (F.W. = 121.14 g/mol)
2. Fill up to 300 mL of milliQ water and then adjust pH to 8.5.
3. Top off to a final volume of 500 mL with milliQ water.
*When making buffers for coupling the amine-modified oligo to sepharose beads, make sure
to make the Quench Buffer last, as contaminating your other buffers with the Tris that it contains
will likely result in the oligo not binding to the beads properly.
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Buffer HIC - A (DON'T USE THIS BUFFER - THE KCl PRECIPITATES OUT OF SOLUTION)
Components:
1. 20 mM HEPES-KOH, pH 7.2
- 10 mL of 1 M HEPES-KOH, pH 7.2
2. 1 mM MgCl2
- 1 mL of 500 mM MgCl2
3. 3.5 M KCl
- 130.5 grams of KCl powder
4. Top off to a final volume of 500 mL with milliQ water.
*Add DTT to a final concentration of 1 mM right before using the buffer.
- This is the buffer meant to be used for my Hydrophobic Interaction Column purifications of unlabeled
eEF2-YBBR from labeled eEF2-YBBR
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Buffer HIC - A - NaCl
Components:
1. 20 mM HEPES-KOH, pH 7.2
- 10 mL of 1 M HEPES-KOH, pH 7.2
2. 1 mM MgCl2
- 1 mL of 500 mM MgCl2
3. 3 M NaCl
- 87.66 grams of NaCl powder
4. Top off to a final volume of 500 mL with milliQ water.
*Add DTT to a final concentration of 1 mM right before using the buffer.
- This is the buffer meant to be used for my Hydrophobic Interaction Column purifications of unlabeled
eEF2-YBBR from labeled eEF2-YBBR
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Buffer HIC - B
Components:
1. 20 mM HEPES-KOH, pH 7.2
- 10 mL of 1 M HEPES-KOH, pH 7.2
2. 1 mM MgCl2
- 1 mL of 500 mM MgCl2
3. Top off to a final volume of 500 mL with milliQ water.
*Add DTT to a final concentration of 1 mM right before using the buffer.
- This is the buffer meant to be used for my Hydrophobic Interaction Column purifications of unlabeled
eEF2-YBBR from labeled eEF2-YBBR
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