Supplementary Materials: PANOx-SP Premix Protocols
Supplementary Materials
Simplifying and streamlining E. coli-based cell-free protein synthesis
Preparation of a PANOx-SP premix solution for cell-free protein synthesis
1. Introduction
This section details the procedures for the preparation of individual cell-free protein
synthesis (CFPS) precursor solutions and combination of these solutions into 500-mL of a
convenient 4x premix (Figure S1). 500-mL of the 4x premix is sufficient to perform 2L of
CFPS. If volumes smaller or larger volumes than 500-mL are desired, the amount of reagents
and chemicals must be scaled down or up accordingly. The precursor solutions have typically
been prepared using the following protocols in the following order:
1) Preparation of a Magnesium Glutamate Solution
2) Preparation of a 10x Salt Solution
3) Preparation of a 25x Nucleotide Mix
4) Preparation of a 25x 19 amino acid mix
5) Preparation of a 25x PEP solution
6) Testing of individual CFPS precursor solutions (Test batch of 4x Premix)
7) Combination of individual CFPS precursor solutions into a Final 4x Premix solution
Equipment Note: Using a heated stir plate with temperature control along with a pH probe and
pH meter will be helpful in preparing the CFPS precursor solutions and Premix.
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Supplementary Materials: PANOx-SP Premix Protocols
(2) Prepare Test 4x Premix
(1) Prepare precursor solutions,
aliquot, and freeze at -80° C
MgGlu
50µL
10x SS
MgGlu
Magnesium
Glutamate
25x N-Mix
MgGlu
rest
25x AA
250µL
10x Salt
Solution (SS)
Thaw small aliquots, mix,
and make Test 4x Premix
25x PEP
10x SS
Perform CFPS with
Test 4x Premix and
identify problematic
reagents (if any)
10x SS
rest
100µL
25x
Nucleotide
Mix (N-Mix)
25x N-Mix
25x N-Mix
rest
100µL
25x 19 Amino
Acid Mix (AA)
(3) Prepare Final 4x Premix
MgGlu
25x AA
10x SS
25x AA
rest
25x N-Mix
100µL
Thaw large aliquots and
make Final 4x Premix
25x PEP
Aliquot Final 4x Premix,
freeze, and store at -80° C
25x AA
25x PEP
25x PEP
25x PEP
rest
Perform CFPS with
Final 4x Premix to confirm
protein synthesis activity
Figure S1: Flow diagram for preparation of a 4x premix solution for CFPS.
2. Preparation of a Magnesium Glutamate Solution
(The following protocol describes the preparation of 44 mL of 1000 mM magnesium glutamate
solution)
Magnesium glutamate is kept as a separate addition because the magnesium
concentration for maximum protein production varies from one lot of cell extract to another. The
typical optimal magnesium glutamate concentration for PANOx-SP is in between 10-20 mM.
The default magnesium glutamate for the preparation described in this work is 10 mM.
Therefore, additional magnesium glutamate may need to be added to reach the optimum
concentration depending on the particular extract that is used.
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Supplementary Materials: PANOx-SP Premix Protocols
Reagent
L-Glutamic
Acid,
Hemimagnesium
Tetryhydrate
(Magnesium
Glutamate)
Formula
Weight
(g/mol)
388.6
Main
Precursor
Concentration
1000 mM
Concentration
in CFPS
Required
Quantity (g)
TBD
17.1
Actual
Quantity (g)
Table S1: 1000 mM Magnesium Glutamate Component.
1) Determine the desired final volume of the magnesium glutamate solution based on the
mass (44 mL). Determine the required quantity of magnesium glutamate (17.1 g).
2) Obtain an appropriately sized mixing vessel and place it on an Isotemp stir plate (Fisher).
3) Place a stir bar in the mixing vessel and set the heating temperature to 37 °C.
4) Add autoclaved MQ water to the vessel (75% of the final volume).
5) Set the stir rate to a high mixing rate, but avoid splashing.
6) Weigh out the required quantity of magnesium glutamate in a weigh boat and pour the
quantity into the mixing vessel.
7) Allow the magnesium glutamate to dissolve at 37 °C for 10 minutes with stirring.
8) Determine the volume of the solution using a pipette or graduated cylinder.
9) Add autoclaved MQ water to obtain the desired final volume. Allow the solution to stir at
room temperature for 1 – 2 minutes until fully homogeneous.
10) Determine the pH of the solution and record the value. The pH should be approximately
6.4 - 6.5.
11) Aliquot 50 µL into an eppendorf tube for subsequent reagent testing.
12) Aliquot the remaining solution into 50-mL conical tubes, flash freeze using liquid
nitrogen and store at -80 °C.
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Supplementary Materials: PANOx-SP Premix Protocols
3. Preparation of the 10x Salt Solution
(The following protocol describes the preparation of 220 mL of 10x Salt Solution)
Reagent
L-Glutamic
Acid,
Monopotassium
Salt (Potassium
Glutamate)
L-Glutamic
Acid,
Monopotassium
Salt
(Ammonium
Glutamate)
Potassium
Oxalate,
Monohydrate
Formula
Weight
(g/mol)
203.2
Main
Precursor
Concentration
1750 mM
Concentration
in CFPS
Required
Quantity (g)
175 mM
78.3
164.2
100 mM
10 mM
3.6
184.2
27 mM
2.7 mM
1.1
Actual
Quantity (g)
Table S2: 10x Salt Solution Components.
1) Determine the desired final volume of the 10x salt solution (220 mL). Determine the
required quantity of potassium glutamate, ammonium glutamate, and potassium oxalate
(Table S2 provides the required masses to prepare 220 mL of a 10x Salt Solution).
2) Obtain an appropriately sized mixing vessel and place it on the stir plate. Place a stir bar
in the mixing vessel.
3) Add autoclaved MQ water (45% of the desired final volume). Set the stir rate to a high
mixing rate, but avoid splashing. Set the stir plate heating to 37 °C.
4) Weigh out the required quantity of potassium glutamate and add into the beaker.
Subsequent reagents can be added immediately.
5) Weigh out the required quantity of ammonium glutamate and add into the beaker.
6) Weigh out the required quantity of potassium oxalate and pour into the beaker.
7) Allow the salts to dissolve at 37 °C for 10 minutes on the stir plate.
8) Determine the volume of the solution using a pipette or graduated cylinder.
9) Add autoclaved MQ water to obtain the desired final volume.
10) Allow the salts to dissolve at room temperature. This may take 10 minutes.
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Supplementary Materials: PANOx-SP Premix Protocols
11) Obtain a pH reading for the solution once all components are completely dissolved. The
pH should be approximately 7.2.
12) Aliquot 250 µL for subsequent reagent testing.
13) Aliquot the remaining solution into multiple 50 mL falcon tubes, flash freeze with liquid
nitrogen, and store at -80 °C.
4. Preparation of a 25x Nucleotide Mix
(The following protocol describes the preparation of 88 mL of 25x Nucleotide Mix)
The Nucleotide Mix contains polycations, cofactors, and the nucleotide triphosphates and
monophosphates required for cell-free protein synthesis. The polycations putrescine and
spermidine are difficult to weigh out as solids, so concentrated precursor solutions must be
prepared prior to making the 25x Nucleotide Mix. The procedures to do this are detailed in
sections 5 and 6. Appropriate volumes of the concentrated precursor solutions are then added
along with the appropriate masses of the other chemical components to form the 25x Nucleotide
Mix, detailed in section 7.
Reagent
Formula
Weight
(g/mol)
Concentration
in CFPS
Required Volume
(mL) of Precursor
Solution
88.2
Desired
Concentration
in 25x
Nucleotide
Mix
25 mM
1000 mM 1,4Diaminobutane
Solution
(Putrescine)
1500 mM
Spermidine
Solution
Reagent
1 mM
2.2
145.2
37.5 mM
1.5 mM
2.2
Formula
Weight
(g/mol)
Concentration
in CFPS
Required Mass (g)
663.4
Desired
Concentration
in 25x
Nucleotide
Mix
8.3 mM
Nicotinamide
Adenine
Dinucleotide
(NAD)
Adenosine 5’Triphosphate
Disodium Salt
(ATP)
0.33mM
0.5
551.1
30 mM
1.2 mM
1.5
5
Actual Volume
(mL) of
Precursor
Solution
Actual Mass (g)
Supplementary Materials: PANOx-SP Premix Protocols
Cytidine 5′monophosphate
disodium salt
(CMP)
Guanosine 5′monophosphate
disodium salt
hydrate (GMP)
Uridine 5′monophosphate
disodium salt
(UMP)
Coenzyme A
Hydrate (CoA)
MRE600 E.coli
tRNA (tRNA)
Folinic Acid,
Calcium Salt
367.16
21.5 mM
0.86 mM
1.0
407.18
21.5 mM
0.86 mM
1.0
368.15
21.5 mM
0.86 mM
1.0
767.5
6.8 mM
0.27 mM
0.5
N/A
4.3 mg/mL
170 µg/mL
0.4
511.5
0.9 mg/mL
34 µg/mL
0.1
Table S3: 25x Nucleotide Mix Components.
5. Preparation of a 1000 mM 1,4-diaminobutane (Putrescine) precursor solution
(The following protocol describes the preparation of 284 mL of 1000 mM putrescine from a 25.0
g bottle of 1,4-diaminobutane, otherwise known as putrescine)
1) Determine the final volume for the 1000 mM 1,4-Diaminobutane precursor solution
based on the mass (284 mL).
2) Obtain a 400 mL beaker and place it on ice. Place a stir bar in the beaker.
3) Add 20 mL autoclaved MQ water directly to the bottle of 1,4-diaminobutane.
4) Vortex the bottle vigorously for 1-2 minutes to facilitate dissolution. Pour out the liquid
from the bottle into the beaker.
5) Repeat steps 3 and 4 two times. After adding the final 20 mL of Autoclaved MQ water,
let the water in the bottle sit on ice for 10 – 15 minutes.
6) Pour out the liquid from the bottle into the beaker. At this point the 1,4-diaminobutane
should be completely dissolved or the solid should have dislodged from the bottle. As
needed, more water can be added to dissolve any remaining reagent. However, do not
exceed the desired final volume.
7) Once the 1,4-Diaminobutane has dissolved, rinse out the bottle with 10 mL of autoclaved
MQ water and pour it into the beaker.
8) Determine the volume of the solution using a pipette or graduated cylinder.
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Supplementary Materials: PANOx-SP Premix Protocols
9) Add autoclaved MQ water to obtain the desired final volume. Place the beaker onto a stir
plate. Allow the solution to stir at room temperature for 1 - 2 minutes.
10) The pH should be approximately 13.
11) Leave the solution on ice for immediate use or aliquot in multiple 50-mL conical tubes.
If you choose to aliquot, then flash freeze using liquid nitrogen and store at -80 °C.
6. Preparation of a 1500 mM Spermidine precursor solution
(The following protocol describes the preparation of 23 mL of 1500 mM spermidine from a 5.0 g
bottle of spermidine)
1) Determine the final volume for the 1000 mM spermidine precursor solution based on the
mass (23 mL).
2) Obtain a beaker and place it onto a stir plate with a stir bar.
3) Add 5 mL autoclaved MQ water to the bottle of spermidine.
4) Shake/vortex the bottle vigorously for 1 – 2 minutes to facilitate dissolution. Pour out the
liquid from the bottle into the mixing vessel.
5) Repeat steps 3 and 4.
6) Once all of the spermidine has dissolved rinse out the bottle with autoclaved MQ water
and pour it into the mixing vessel.
7) Determine the volume of the solution using a pipette or graduated cylinder.
8) Add autoclaved MQ water to obtain the desired final volume. Allow the solution to stir at
room temperature for 1 – 2 minutes until fully homogeneous.
9) The pH of the solution should be approximately 13.
10) Leave the solution on ice for immediate use or aliquot in 50-mL conical tubes. If you
choose to aliquot, then flash freeze using liquid nitrogen and store at -80 °C.
7. Preparation of the 25x Nucleotide Mix using the Putrescine and Spermidine precursor
solutions
1) Determine the desired final volume for the 25x Nucleotide Mix solution (88 mL).
Determine the required quantity of each of the liquid and solid reagents (Table S3
provides the required volumes and masses for preparing 88 mL of a 25x Nucleotide Mix).
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Supplementary Materials: PANOx-SP Premix Protocols
2) Obtain an appropriately sized mixing vessel and place it onto a stir plate. Place a stir bar
into the bottom of the mixing vessel.
3) Add 50% of the final volume, autoclaved MQ water to the vessel.
4) Set the stir rate to a sufficient mixing rate and such that splashing does not occur.
5) Add the required volume of the 1000 mM putrescine precursor solution to the mixing
vessel.
6) Add the required volume of the 1500 mM spermidine precursor solution to the mixing
vessel.
7) Weigh out the required quantity of NAD in a weigh boat and pour the quantity into the
beaker. If lyophilized NAD in 50 mg vials are used, add 1mL of autoclaved MQ water to
each vial that is needed. Vortex until fully dissolved. Add the appropriate volume of
NAD solution. Assume that the contents of each vial, once dissolved, contains 50 mg of
NAD.
8) Ensure the complete dissolution of the material before proceeding with the next step.
9) Add the required quantity of CoA into the beaker.
10) Ensure the complete dissolution of the material before proceeding with the next step.
11) Add the required quantity of to the beaker.
12) Ensure the complete dissolution of the material before proceeding with the next step.
13) Add the required quantity of ATP into the beaker. If whole vials of ATP are used, 1 mL
of water may be used to rinse out each of the emptied vial, previously containing 1 g
ATP.
14) Add CMP, GMP, and UMP in that order, in a fashion similar to the ATP described
above. Ensure the complete dissolution of the material before proceeding with the next
step.
15) Add the required quantity of Folinic Acid in a weigh boat and pour the quantity into the
beaker.
16) Determine the volume of the solution using a pipette or graduated cylinder. Record the
quantity.
17) Add autoclaved MQ water to obtain the desired final volume.
18) Determine the pH of the solution and record the value. The pH should be approximately
7.4 - 7.6.
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Supplementary Materials: PANOx-SP Premix Protocols
19) Aliquot 100 µL for reagent testing. Aliquot the remaining solution into multiple 50-mL
conical tubes, flash freeze with liquid nitrogen, and store at -80 °C.
8. Preparation of 25x 19 Amino Acid Mix (50mM of each amino acid)
(The following describes the preparation of 200 mL of 25x 19 Amino Acid Mix)
This is the precursor solution of amino acids that will be used by the CFPS reaction.
Glutamate is omitted from this precursor because it will already be present in the form of
potassium glutamate in the previously prepared 10x Salt Solution.
Order of Addition /
Reagent
Formul
a
Weight
(g/mol)
Quantity (g)
pH After Addition
Dissoluti
on Time
(sec)
Requir
ed
Approx.
Approx.
Actual
Actual
1
L-Arginine
174.2
1.7420
10.65 –
10.68
30
2
L-Valine
117.1
1.1710
8.99 – 9.20
180
3
L-Tryptophan
204.2
2.0420
8.66 – 8.71
300
4
LPhenylalanine
165.2
1.6520
8.42 – 8.46
300
5
L-Isoleucine
131.2
1.3120
8.31 – 8.34
480
6
L-Leucine
131.2
1.3120
8.22 – 8.25
480
7
L-Cysteine
121.2
1.2120
7.80 – 7.84
120
8
L-Methionine
149.2
1.4920
7.73 – 7.75
240
9
DL-Alanine
89.09
0.8909
7.76 – 7.77
30
10
L-Asparagine
132.1
1.3210
7.64 – 7.71
60
11
L-Aspartic
Acid
133.1
1.3310
5.35 – 5.41
180
12
L-Glycine
75.07
0.7507
5.34 – 5.42
60
13
L-Glutamine
146.1
1.4610
5.34 – 5.40
120
14
L-Histidine
155.2
1.5520
6.46 – 6.54
240
15
L-Lysine
Monohydroch
loride
182.6
1.8260
6.47 – 6.54
60
16
L-Proline
115.1
1.1510
6.49 – 6.47
120
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Supplementary Materials: PANOx-SP Premix Protocols
17
DL-Serine
105.1
1.0510
6.49 – 6.47
30
18
L-Threonine
119.1
1.1910
6.50 – 6.54
30
19
L-Tyrosine
181.2
1.8120
NA
NA
Table S4: 25x (50 mM) 19 Amino Acid Mix Components.
1. Obtain each of the reagents and set up the stir plate. Place a stir bar into a 300 mL beaker.
Add 87.5% (175 mL) of the final solution volume, autoclaved MQ water to the beaker.
2. Place the temperature probe into the water close to the edge of the beaker about half way into
the water (without having the probe touching the beaker). Set the heating temperature to 37
°C. Allow the water to reach temperature. Elevated temperatures at even 45 °C for extended
time will decrease solution activity.
3. Set the stir rate to 600 rpm. If the final volume is being scaled up, the stir rate should be set
so that the bottom of the vortex forms just above the stir bar.
4. Weigh out the required quantity of each of the reagents in Table S4. Add each of the
reagents, one at a time according to the order of addition in Table S4. After dissolution of
each reagent, measure the pH. Do not add the subsequent reagent until the previous is fully
dissolved. Use the “approx. dissolution time” and “approx. pH” as a guide. Repeat this step
until tyrosine addition. After threonine has dissolved, allow the solution to mix for an
additional 5 minutes.
5. Turn off heating on the heating/stir plate.
6. Pour the solution into a graduated cylinder to determine the volume. Add autoclaved MQ
water to bring the total volume to 200 mL (approximately 5% of the final volume will be
needed). Sterile filtering the solution may decrease the activity of the final mixture by
removing undissolved amino acids. The solution should appear clear and colorless and
relatively free of particulates. Freezing the solution at this point should not cause any
precipitation.
7. Pour solution back into the beaker and turn on stirring to 600 rpm. While mixing, add the
required quantity of tyrosine to the solution and allow the suspension to mix for 5 minutes.
Note: the tyrosine will not fully dissolve and will remain in suspension.
8. While still mixing, aliquot 100µL for subsequent reagent testing.
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Supplementary Materials: PANOx-SP Premix Protocols
9. While still mixing, aliquot the remaining solution into multiple 50-mL conical tubes. Flash
freeze the aliquots using liquid nitrogen and store in the -80 °C freezer.
9. Preparation of a 25x PEP Solution (833 mM PEP)
(The following protocol describes the preparation of 81.5 mL of 833 mM PEP)
Reagent
Phosphoenolypyruate
(PEP)
Formula
Weight
(g/mol)
206
Desired
Concentration
in 25x Mix
833 mM
Concentration
in CFPS
Required Mass
(g)
33 mM
14
Actual Mass
(g)
Table S5: 25x PEP Solution Component.
1) Obtain a 200 mL beaker and place it onto a stir plate. Place a stir bar in the beaker. Set up
the pH probe by placing it close to the side of the beaker without it touching the walls or
the stir bar.
2) Determine the final desired volume for the 25x PEP (81.5 mL). Determine the required
amount of PEP for the desired volume (14 g).
3) Add the required quantity of PEP into the beaker. If possible, try to keep the PEP chilled
on ice. The PEP should not be left out at room temperature for an extended period of
time.
4) For each 1g of PEP used, add 1 mL of water to the beaker. If more than one vial of PEP
is used, pour the PEP into the mixing vessel. Then use the 1 mL of water to rinse out any
empty bottles of PEP. At this point, the PEP should be mostly insoluble. Increasing the
pH will be required to dissolve PEP.
5) Adjust the pH of the PEP solution using 10 M KOH by placing the pH probe into PEP
solution. Slowly add 10 M KOH until the pH of the solution reaches pH 7.3 ±0.1 at 22 –
25 °C. The pH increases sharply as the pH nears 7.3 (see attached PEP titration curve,
Figure S2). Note: target pH = 7.4.
6) Ensure the complete dissolution of the material before proceeding.
7) Determine the new volume of PEP precursor solution.
8) Determine the volume of the solution using a pipette or graduated cylinder.
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Supplementary Materials: PANOx-SP Premix Protocols
9) Add autoclaved MQ water to obtain the desired volume of 25x PEP solution.
10) Aliquot 100 µL for subsequent reagent testing.
11) Aliquot the remaining solution into multiple 50-mL conical tubes, flash freeze with liquid
nitrogen, and store at -80 °C.
PEP Titration (5g PEP / 5mL Water)
PEP Titration
8
7
6
pH
5
4
3
2
1
0
0
1000
2000
3000
4000
5000
6000
Volume Titrant (uL)
Figure S2: Example PEP titration curve.
10. Testing of individual CFPS precursor solutions
All of the above reagents must be first tested at small scale to ensure that all the
components are of sufficient quality. Problematic reagents (if any) must be identified and
replaced.
Component
a. Autoclaved MQ Water
b. Magnesium Glutamate
c. 10x Salt Solution Mix
d. 25x Nucleotide Mix
e. 25x 19 Amino Acid Mix
f. 25x PEP
Required Quantity (uL)
= 500 – (a + b + c + d + e + f)
20 (For 10 mM)
200
80
80
80
Table S6: Preparation of 500 µL of Test 4x Premix.
1) Thaw the small aliquots (eppendorf tubes) of the precursor solutions.
2) Using the small aliquots, create a scale-down Test 4x Premix solution at 500 µL. This
will be used to test the quality of precursor solutions.
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Supplementary Materials: PANOx-SP Premix Protocols
3) Pipette the required volume of autoclaved MQ water into an eppendorf tube.
4) Add the required volume of the 1000 mM Magnesium Glutamate solution into the
eppendorf tube.
5) Add the required volume of the 10x Salt Solution into the eppendorf tube.
6) Add the required volume of the 25x Nucleotide Mix solution into the eppendorf tube.
7) Add the required volume of the 25x 19 Amino Acid Mix into the eppendorf tube.
8) Add the required volume of the 25x PEP solution into the eppendorf tube.
9) Vortex the solution so that it is homogeneous.
10) Perform a set of cell-free reactions using plasmid encoding for chloramphenicol acetyl
transferase (CAT) to test reagents. The average total protein yield is between 800-1000
µg/mL.
11) Once the yields have been determined to be sufficient, proceed to the next section of the
protocol. If the yields are not sufficient, troubleshooting will be required to determine
which components are responsive for the low protein yields. A magnesium optimization
may need to be performed for each extract.
11. Combination of individual CFPS precursor solutions into a Final 4x Premix solution
Table S7 describes the preparation of the Final 4x PANOx-SP premix. Once the quality
of the precursor solutions has been verified using the Test 4x Premix, the large aliquots of the
precursor solutions can then be mixed to form the Final 4x Premix. The CFPS yields using the
Final 4x Premix should be similar to that of the Test 4x Premix.
Component
a. Autoclaved MQ Water
b. Magnesium Glutamate
c. 10x Salt Solution Mix
d. 25x Nucleotide Mix
e. 25x 19 Amino Acid Mix
f. 25x PEP
Required Quantity (mL)
= 500 – (a + b + c + d + e + f)
20 (For 10 mM)
200
80
80
80
Table S7: Preparation of 500mL of Final 4x Premix.
1) Thaw the large aliquots (50-mL conical tubes) of the precursor solutions.
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Supplementary Materials: PANOx-SP Premix Protocols
2) Obtain a clean 1000 mL beaker and place it onto a stir plate. Place a stir bar into the
bottom of the mixing vessel along with the required amount of autoclaved MQ water. Set
the stir rate to a sufficient mixing rate, such that splashing does not occur.
3) Add the required volume of the 1000 mM Magnesium Glutamate solution into the
beaker. This value will be dependent on the concentration of magnesium in the cell
extract.
4) Add the required volume of the 10x Salt Solution into the beaker.
5) Add the required volume of the 25x Nucleotide Mix solution into the beaker.
6) Add the required volume of the 25x 19 Amino Acid Mix into the beaker.
7) Add the required volume of the 25x PEP solution into the beaker.
8) Increase the stir rate to setting 4.
9) Aliquot the premix into the appropriate 50 µL to 50 mL quantities as desired.
10) Flash freeze the premix aliquots using liquid nitrogen and store at -80 °C.
11) Flash freeze the remaining quantities of 1000 mM Magnesium Glutamate, 10x Salt
Solution, 25x Nucleotide Mix, 25x 19 Amino Acid Mix, and 25x PEP solution and store
at -80 °C.
12. Conclusions
Using the above protocol, we can conveniently produce 500 mL of 4x CFPS premix
solution for rapid, convenient, and high yield production of a variety of proteins using cell-free
protein synthesis.
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