TITLE: S30-S30A-S30-Buffers- Haseloff Lab
AUTHORS: Fernando Guzman Chavez, Jim Haseloff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:center"><span>Following this recipe, you will obtain 1L of</span><span style = "font-weight:bold;"> S30</span><span style = "font-weight:bold;font-weight:bold;">,</span><span style = "font-weight:bold;"> S30A </span><span>and</span><span style = "font-weight:bold;"> S30B Buffers</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">S30 Buffer </span><span>is a standard solution to wash cell pellets previous to lysis. Likewise, it is used during the cell lysis.</span></div></div><div class = "text-block">NOTE :</div><div class = "text-block">The protocol described here is an adaptation from these papers:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">Adam D. Silverman</span><span>, Nancy Kelley-Loughnane, Julius B. Lucks, and Michael C. Jewett (2019).</span><span style = "font-style:italic;">Deconstructing Cell-Free Extract Preparation for in Vitro Activation of Transcriptional Genetic Circuitry</span><span>. ACS Synthetic Biology, 403-414. DOI: 10.1021/acssynbio.8b00430.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">Yang WC,</span><span> Patel KG, Wong HE, Swartz JR.(2012). </span><span style = "font-style:italic;">Simplifying and streamlining Escherichia coli-based cell-free protein synthesis</span><span>.Biotechnol Prog. 28(2):413-420. DOI:10.1002/btpr.1509.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;">https://www.protocols.io/researchers/anibal-arce-medina</li></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">Yang WC,</span><span> Patel KG, Wong HE, Swartz JR.(2012). </span><span style = "font-style:italic;">Simplifying and streamlining Escherichia coli-based cell-free protein synthesis</span><span>.Biotechnol Prog. 28(2):413-420. DOI:10.1002/btpr.1509.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;">https://www.protocols.io/researchers/anibal-arce-medina</li></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div></div>

[STEPS]
?. [1. S30 Buffer]
To prepare S30 buffer, following compounds have to be ready-to-use:Tris-AcetateMagnesium acetate tetrahydratePotassium acetate1M DTT5M KOH
?. [1. S30 Buffer]
1. 1 Prepare for autoclavingVolumes indicated are sufficient for 1 L of S30 buffer 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.  ABCD1Compound, Sum FormulaMW [g/mol]Required amount [g or mL]Concentration [mM]2Tris-Acetate 181.19 1.81g103Magnesium acetate214.45 3.0g144Potassium  acetate98.145.89g60Before 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.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.
ABCD1Compound, Sum FormulaMW [g/mol]Required amount [g or mL]Concentration [mM]2Tris-Acetate 181.19 1.81g103Magnesium acetate214.45 3.0g144Potassium  acetate98.145.89g60
?. [2. S30A and S30B Buffer]
To prepare S30A and S30B buffers, following compounds have to be ready-to-use:100 mM Tris (solution)1:1AceticAcid1M DTT
?. [2. S30A and S30B Buffer]
2.1 Prepare for autoclavingVolumes indicated are sufficient for 1 L of S30 Buffer A and S30 Buffer BS30 Buffer A Before use add 2 mL of 1 M DTTS30 Buffer B Before use add 2 mL of 1 M DTTS30 Buffer A  ABCD1Compound,  Sum FormulaMW [g/mol]Required amount [g or mL]Concentration [g/L]2100 mM Tris Base121.14500 mL6.057 [50 mM]3Mg-Glutamate 388.615.44g5.44 [14mM]4K-Glutamate 203.2312.194g12.194[60mM]Before use add 2 mL of 1 M DTTS30 Buffer B Before use add 2 mL of 1 M DTTS30 Buffer B  ABCD1Compound,  Sum FormulaMW [g/mol]Required amount [g or mL]Concentration [g/L]2100 mM Tris Base121.1450 mL0.6057  [5 mM]3Mg-Glutamate 388.615.44g5.44[14mM]4K-Glutamate 203.2312.194g12.194[60mM]
ABCD1Compound,  Sum FormulaMW [g/mol]Required amount [g or mL]Concentration [g/L]2100 mM Tris Base121.14500 mL6.057 [50 mM]3Mg-Glutamate 388.615.44g5.44 [14mM]4K-Glutamate 203.2312.194g12.194[60mM]
ABCD1Compound,  Sum FormulaMW [g/mol]Required amount [g or mL]Concentration [g/L]2100 mM Tris Base121.1450 mL0.6057  [5 mM]3Mg-Glutamate 388.615.44g5.44[14mM]4K-Glutamate 203.2312.194g12.194[60mM]
?. [3. Literature/ References]
?. [4. Change history]
V1→V1.1 TBST recipe added.V1.1→V1.2 Recipes to prepare S30A and S30B buffers were added.