Jul 11, 2020

Public workspaceS30-S30A-S30-Buffers- Haseloff Lab

DOI
dx.doi.org/10.17504/protocols.io.bigckbsw
  • 1University of Cambridge
Fernando FGC Guzman Chavez
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DOI: dx.doi.org/10.17504/protocols.io.bigckbsw
Protocol Citation: Fernando FGC Guzman Chavez, Jim Haseloff 2020. S30-S30A-S30-Buffers- Haseloff Lab. protocols.io https://dx.doi.org/10.17504/protocols.io.bigckbsw
License: This is an open access protocol 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
Protocol status: Working
We use this protocol and it's working
Created: July 10, 2020
Last Modified: July 11, 2020
Protocol Integer ID: 39140
Keywords: S30 buffer, cell-free,
Abstract
Following this recipe, you will obtain 1L of S30, S30A and S30B Buffers

S30 Buffer is a standard solution to wash cell pellets previous to lysis. Likewise, it is used during the cell lysis.

NOTE :

The protocol described here is an adaptation from these papers:

  • Adam D. Silverman, Nancy Kelley-Loughnane, Julius B. Lucks, and Michael C. Jewett (2019).Deconstructing Cell-Free Extract Preparation for in Vitro Activation of Transcriptional Genetic Circuitry. ACS Synthetic Biology, 403-414. DOI: 10.1021/acssynbio.8b00430.

  • Yang WC, Patel KG, Wong HE, Swartz JR.(2012). Simplifying and streamlining Escherichia coli-based cell-free protein synthesis.Biotechnol Prog. 28(2):413-420. DOI:10.1002/btpr.1509.

  • https://www.protocols.io/researchers/anibal-arce-medina


1. S30 Buffer
1. S30 Buffer
To prepare S30 buffer, following compounds have to be ready-to-use:

  • Tris-Acetate
  • Magnesium acetate tetrahydrate
  • Potassium acetate
  • 1M DTT
  • 5M KOH
1. 1 Prepare for autoclaving

  • Volumes indicated are sufficient for 1 L of S30 buffer



Compound, Sum FormulaMW [g/mol]Required amount [g or mL]Concentration [mM]
Tris-Acetate 181.19 1.81g10
Magnesium acetate214.45 3.0g14
Potassium acetate98.145.89g60
Preparation: Dissolve in demi-H2O, fill up to 1000 ml, autoclave 15 min at 121°C
*adjust to pH=8.2 with 5M KOH (approx. 900µL)

  • Before useadd 2 mL of 1 M DTT(2mM, final concentration)

  • For dialysis, S30 buffer is prepared at 5mM Tris-Acetate and 1mM DTT. Rest of the components remain at same concentrations.

  • To prepareTBST Buffer (pH=7.6), dissolve 1 tablet of Tris buffered saline with Tween 20 in 500mL of water and autoclave 15 min at 121°C. Catalogue Number: 91414-10TAB SIGMA.
2. S30A and S30B Buffer
2. S30A and S30B Buffer
To prepare S30A and S30B buffers, following compounds have to be ready-to-use:

  • 100 mM Tris (solution)
  • 1:1AceticAcid
  • 1M DTT

2.1 Prepare for autoclaving

  • Volumes indicated are sufficient for 1 L of S30 Buffer A and S30 Buffer B

S30 Buffer A

Compound, Sum FormulaMW [g/mol]Required amount [g or mL]Concentration [g/L]
100 mM Tris Base121.14500 mL6.057 [50 mM]
Mg-Glutamate 388.615.44g5.44 [14mM]
K-Glutamate 203.2312.194g12.194[60mM]
Dissolve in demi-H2O, fill up to 1000 ml, autoclave 15 min at 121°C
*adjust to pH=7.7 with acetic acid

  • Before use add 2 mL of 1 M DTT

S30 Buffer B


Compound, Sum FormulaMW [g/mol]Required amount [g or mL]Concentration [g/L]
100 mM Tris Base121.1450 mL0.6057  [5 mM]
Mg-Glutamate 388.615.44g5.44[14mM]
K-Glutamate 203.2312.194g12.194[60mM]
Dissolve in demi-H2O, fill up to 1000 ml, autoclave 15 min at 121°C
*adjust to pH=8.2 with acetic acid


  • Before use add 2 mL of 1 M DTT
3. Literature/ References
3. Literature/ References

CITATION
Silverman AD, Kelley-Loughnane N, Lucks JB, Jewett MC (2019). Deconstructing Cell-Free Extract Preparation for in Vitro Activation of Transcriptional Genetic Circuitry.. ACS synthetic biology.
LINK
https://doi.org/10.1021/acssynbio.8b00430

CITATION
Yang WC, Patel KG, Wong HE, Swartz JR (2012). Simplifying and streamlining Escherichia coli-based cell-free protein synthesis.. Biotechnology progress.
LINK
https://doi.org/10.1002/btpr.1509
  • https://www.protocols.io/researchers/anibal-arce-medina
4. Change history
4. Change history
V1→V1.1 TBST recipe added.
V1.1→V1.2 Recipes to prepare S30A and S30B buffers were added.
Citations
Step 3
Silverman AD, Kelley-Loughnane N, Lucks JB, Jewett MC. Deconstructing Cell-Free Extract Preparation for in Vitro Activation of Transcriptional Genetic Circuitry.
https://doi.org/10.1021/acssynbio.8b00430
Step 3
Yang WC, Patel KG, Wong HE, Swartz JR. Simplifying and streamlining Escherichia coli-based cell-free protein synthesis.
https://doi.org/10.1002/btpr.1509