CELL-FREE PROTEIN SYNTHESIS KIT (PSS3050)
TRANSLATION REAGENT SET (TLS4050)
PROTOCOL

Aug, 2018 / V1.7
Prohibited to reprint all or part of this document without permission

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INTRODUCTION
Protein synthesis using cell-free protein synthesis reagent kit consists of three stages: preparationof transcription template, transcription and translation. For the use of the translation reagent set,please refer to section “Translation”.

In preparation of transcription template, it is necessary to prepare a primer in advance.
In the different attachment, five different tag sequences are shown, and by carrying out PCR twice,
it is possible to efficiently create a transcription template containing multiple tag sequences. Also, it ispossible to tag both N-terminal and C-terminal. In addition, this protocol and primer list can be
downloaded from our website (https://nuprotein.jp/knowledge-base/).
N-term tagged template
5'UTR

Translated Region

3'UTR

86 mer75 merATG

T7 promoter

OMEGA
sequence

ATG

Tag sequence

Cap

Linker

Stop
Tail

ORF

sequence

18 merC-term tagged template
5'UTR

Translated Region

3'UTR

86 mer75 merATG

T7 promoter

OMEGA
sequence

Stop
ORF

LinkerCap

Tag sequence

Tailsequence

21 merIn this protocol, we show the PCR program when used Toyobo Co., Ltd. KOD®-Plus-Neo,
Takara Bio Inc. PrimeSTAR®, PrimeSTAR® Max as PCR enzymes.

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PRIMER DESIGNSTRUCTURE OF N-TERMINAL TAG DNA TEMPLATE
The structure of N-terminal Tag DNA template and the sequence of each part are shown in (Fig.1)
and (Table 1).
Primer design and PCR are more complicated than usual because of the long sequence added tothe prepared ORF.

( Figure.1 )Structure of N-terminal Tag
DNA Template
5'UTR

Translated Region

3'UTR

86 mer75 merATG

T7 promoter

ATG

OMEGA

Tag

sequence

sequence

LinkerCap( Table.1 )RegionORF

Tailsequence

18 merSequences / Part
Site
Cap

5'UTR

Stop

T7 promoter

Sequence

5' to 3'Tag

CCCGC GAAATTAATA CGACT CACTA TAGGGOMEGACTCAC CTATC TCTCT ACACA AAACA TTTCC CTACA TACAA CTTTC

sequence

AACTT CCTAT T
ATG

Tag sequence
Translated

6 x HisGAC TAC AAG GAT GAC GAT GAC AAGFLAGTAT CCT TAT GAC GTG CCT GAC TAT GCC AGC CTG GGA GGA CCT

HA

GGC CTG AAC GAC ATC TTC GAG GCC CAG AAG ATC GAG TGG CAC

RegionGAA
Linker

CTC CAG CAG GGA GGT ACT ( Leu-Gln-Gln-Gly-Gly-Thr )ORF

ATG + NNNNN ・・・ ・・・NNNNN

Stop codon
3'UTR

CAT CAT CAT CAT CAT CAT

Tail sequence

BiotinTAA, TAG, TGA
AAAAA AAAAA GAGCT CTTGG ATCCG GCCAT AAGGT TGGAT CCGGCCATAA GGGCC TGATC CTTCG AGGGG GGGCC

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PROCEDURE FOR PREPARATION
TRANSCRIPTION TEMPLATEOF

PRIMERFOR

N-TERMINAL

TAG

Since PCR is carried out in two stages, 1st PCR and 2nd PCR, multiple primers are required.
(Figure 2).
The Primer List (e.g His-tag insertion) is as shown in (Table 2). Tag refferred in red in (Table 2) can
be change to other Tag .
Please note that Reverse primer is complementary to the sequence of (Table.1).
In the case of non-Tag, please leave the Linker as it is.
The Linker sequence can be changed to an appropriate protease recognition sequence.
( Figure.2 )PCR of N-terminal tag DNA template
2nd-T7-NF2→
ATG

2nd-protein tag-NF1→
1st-gene-specific-NF

→

5'UTR

Translated Region

3'UTR

86 mer75 merATG

T7 promoter

OMEGA
sequence

ATG

Tag sequence

Cap

Linker

Stop
Tail

ORF

sequence

18 mer

1st-gene-specific-NR←
2nd-SU-NR1←
2nd-SU-NCR2←

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( Table.2 )Primer List / N-term His-tag Template DNA
1st PCR

primer type/nameprimer form

1st-gene-specific-NF

Gene specific 1st Fw primer

1st-gene-specific-NR

Gene specific 1st Rv primer

primer sequence

5' to 3'CCAGC AGGGA GGTAC T＋an array of 18 mer at 5' end that startfrom ATG
CCTTA TGGCC GGATC CAAGA GCTCT TTTTT TTTTT TA + an array of
18 mer at 3' end excluding stop codon

2nd PCR
primer type/nameprimer form

2nd-T7-NF2

Universal 2nd Fw primer

primer sequence

5' to 3'CCCGC GAAAT TAATA CGACT CACTA TAG
CGACT CACTA TAGGG CTCAC CTATC TCTCT ACACA AAACA TTTCC

2nd-protein tag-NF1

2nd Fw primer-tag

CTACA TACAA CTTTC AACTT CCTAT TATGC ATCAT CATCA TCATC
ATCTC CAGCA GGGAG GTACT ATG

2nd-SU-NCR2
2nd-SU-NR1

Universal 2nd Rv primer

GGCCC CCCCT CGAAG G
CCCTC GAAGG ATCAG GCCCT TATGG CCGGA TCCAA

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STRUCTURE OF C-TERMINAL TAG DNA TEMPLATE
The structure of C-terminal Tag DNA template and the sequence of each part are shown in (Fig.3)and (Table 3).
Same as with N-terminal tag, Primer design and PCR are more complicated than usual because of
the long sequence added to the prepared ORF( Figure.3 )C-terminal tag DNA template
5'UTR

Translated Region

3'UTR

86 mer75 merATG

T7 promoter

OMEGA
sequence

Stop
ORF

Linker

Cap

Tag

Tail

sequence

sequence

21 mer( Table.3 )Sequences / Part
Region

Site
Cap

5'UTR

T7 promoter

5' to 3'Tag

CCCGC GAAATTAATA CGACT CACTA TAGGGOMEGACTCAC CTATC TCTCT ACACA AAACA TTTCC CTACA TACAA CTTTC

sequence

AACTT CCTAT T

ORF

ATG + NNNNN ・・・ ・・・NNNNN

LinkerGGT CTC CAG CAG GGA GGT ACT ( Gly-Leu-Gln-Gln-Gly-Gly-Thr )Translated
RegionSequence

Tag sequence

CAT CAT CAT CAT CAT CAT

6 x HisGAC TAC AAG GAT GAC GAT GAC AAGFLAGTAT CCT TAT GAC GTG CCT GAC TAT GCC AGC CTG GGA GGA CCT

HA

GGC CTG AAC GAC ATC TTC GAG GCC CAG AAG ATC GAG TGG CAC
GAA
Stop codon
3'UTR

Tail sequence

BiotinTAA, TAG, TGA
AAAAA AAAAA GAGCT CTTGG ATCCG GCCAT AAGGT TGGAT CCGGCCATAA GGGCC TGATC CTTCG AGGGG GGGCC

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PROCEDURE FOR PREPARATION OF PRIMER FOR C-TERMINAL TAG TRANSCRIPTIONTEMPLATE
Since PCR is carried out in two stages, 1st PCR and 2nd PCR, multiple primers are required.
(Figure 4).
The Primer List (i.e. His-tag insertion) is shown in (Table 4). Tag in red (Table.3) can be changed toother Tag sequences.
Please note that Reverse primer is complementary to the sequence of (Table.3)
In the case of non-Tag, please leave the Linker as it is.
The Linker sequence can be changed to an appropriate protease recognition sequence.
( Figure.4 )PCR of C-terminal tag DNA template
2nd-T7-CF→
1st-gene-specific-CF

→

5'UTR

Translated Region

3'UTR

86 mer75 merATG

T7 promoter

OMEGA
sequence

Stop
ORF

LinkerCap

Tail

Tag sequence

sequence

21 mer

1st-gene-specific-CR←
2nd-protein tag-CR1←
2nd-SU-NCR2←

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( Table.4 )Primer List / C-term His-tag Template DNA
1st PCR
primer type/nameprimer form

1st-gene-specific-CF

Gene specific 1st Fw primer

1st-gene-specific-CR

Gene specific 1st Rv primer

primer sequence

5' to 3'

CACAA AACAT TTCCC TACAT ACAAC TTTCA ACTTC CTATT＋an arrayof 18 mer at 5' end that start from ATG
AGTAC CTCCC TGCTG GAGAC C＋an array of 18 mer at 3' end
excluding stop codon

2nd PCR
primer type/nameprimer form

2nd-T7-CF

Universal 2nd Fw primer

2nd-SU-NCR2

Universal 2nd Rv primer

2nd-protein tag-CR1

2nd Rv primer-tag

primer sequence

5' to 3'CCCGC GAAAT TAATA CGACT CACTA TAGGG CTCAC CTATC TCTCTACACA AAACA TTTCC
GGCCC CCCCT CGAAG G
CCCTC GAAGG ATCAG GCCCT TATGG CCGGA TCCAA GAGCT CTTTT
TTTTT TTTAA TGATG ATGAT GATGA TGAGT ACCTC CCTGC TGG

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PRODUCTION OF TRANSCRIPTION TEMPLATE

DETECTION OF 1st PCR AND 2nd PCR PRODUCTPlease check the PCR products after 1st PCR and 2nd PCR as follows.
1.

Prepare a TAE agarose gel at a concentration according to the DNA size and set inelectrophoresis.

2.

Load the 3 μl sample PCR without purification into the agarose gel above.

3.

After completion of electrophoresis, the agarose gel is stained with Ethidium Bromide for 30
minutes.

4.

Fluorescence detection of DNA is performed by a transilluminator.

【Example of checking PCR product for N-terminal tag】

As a result of confirmation of PCR Product, if problem occurs, please review again in thefollowing orderA)
Confirm PCR program
B)
Check composition of the reaction solution preparation
C)
Confirm PCR primer

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1st PCR
The composition and reaction program for one sample are shown in the table below.
It is also recommended to change the description program in consideration of the amplification
efficiency etc.
After completion of 1st PCR reaction, check 1st PCR product (3 μl) using agarose gelelectrophoresis.
In the following program, 1st-gene-specific-F and 1st-gene-specific-R indicate 1st-gene-specific-NF,
1st- gene-specifc-NR in the case of N-terminal tag transcription template, For C-terminal tag
transcription template they are 1st-gene-specific-CF and 1st-gene-specific-CR respectively.

Composition and PCR program when using KOD-Plus-Neo
KOD-Plus-Neo ver.

PCR program
Temp.

Reagents

Time

Cycle

10 X PCR buffer

5 µl

94°C

5 min

1

2 mM dNTPs

5 µl

98°C

10 sec25 mM MgSO4

3 µl

55°C

30 sec

10 µM 1st-gene-specific-F

1 µl

68°C

3 min (1 min/kb)

10 µM 1st-gene-specific-R

1 µl

72°C

2 min

Plasmid

1 ng

20°C

-

KOD

1 µl

Sterile water

X µl

Total

50 µl

30

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Composition and PCR program when using PrimeSTAR
PrimeSTAR ver.

PCR program

Reagents

Temp.

Time

Cycle

5 X PrimeSTAR buffer

10 µl

94°C

5 min

2.5 mM dNTPs

4 µl

98°C

10 sec

10 µM 1st-gene-specific-F

1 µl

55°C

30 sec

10 µM 1st-gene-specific-R

1 µl

68°C

(1 min./kb)

Plasmid

1 ng

72°C

2 min

Polymerase (2.5 U/µl)

0.5 µl20°C-

Sterile water

X µl

Total

50 µl

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Composition and PCR program when using PrimeSTAR Max
PrimeSTAR Max ver.

PCR program

Reagents

Temp.

Time

Cycle

2 X PrimeSTAR Max Premix

25 µl

94°C

5 min

10 µM 1st-gene-specific-F

1 µl

98°C

10 sec

10 µM 1st-gene-specific-R

1 µl

55°C

30 secPlasmid

1 ng

72°C

(5 sec./kb)

Sterile water

X µl

72°C

2 min

Total

50 µl

20°C

-

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2nd PCR
Please use 1st PCR Product in unpurified and unconcentrated state for 2nd PCR
After 2nd PCR reaction, check 2nd PCR product (3 μl) using agarose gel electrophoresis.
It is also recommended to change the description program in consideration of the amplification
efficiency etc.

*Normally, 1 μl of 1st PCR product is used for 2nd PCR.
Each program of 2nd PCR
Following program is common for N-terminal tag and C-terminal tag.

PCR program (KOD or PrimeSTAR ver.)
Temp.

Time

PCR program (PrimeSTAR Max ver.)

Cycle

Temp.

1

98°C

1 min

98°C

10 sec55°C

15 sec

Time

98°C

1 min

98°C

10 sec

60°C

1 min

68°C

3 min

72°C

(5 sec./kb)(1 min/kb)

98°C

10 sec98°C

10 sec60°C

15 sec60°C

15 sec72°C(5 sec./kb)

68°C

3 min

72°C

2 min

20°C

-

10

30

(1 min/kb)
72°C

2 min

20°C

-

Cycle
1

5

30

1

1

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Composition for N-terminal tag of 2nd PCR

KOD-Plus-Neo ver.
Reagents
10 X PCR buffer

5 µl

2 mM dNTPs

5 µl

25 mM MgSO4

3 µl

10 µM 2nd-T7-NF2

1 µl

100 nM 2nd-protein tag-NF1

1 µl

10 µM 2nd-SU-NCR2

1 µl

100 nM 2nd-SU-NR1

1 µl

1st PCR product1-5 µl*KOD

1 µl

Sterile water

X µl

Total

50 µl

PrimeSTAR ver.

Reagents
5 X PrimeSTAR buffer

10 µl

2.5 mM dNTPs

4 µl

10 µM 2nd-T7-NF2

1 µl

100 nM 2nd-protein tag-NF1

1 µl

10 µM 2nd-SU-NCR2

1 µl

100 nM 2nd-SU-NR1

1 µl

1st PCR product1-5 µl*Polymerase (2.5 U/µl)

0.5 µlSterile water

X µl

Total

50 µl

PrimeSTAR Max ver.
Reagents
2 X PrimeSTAR Max Premix

25 µl

10 µM 2nd-T7-NF2

1 µl

100 nM 2nd-protein tag-NF1

1 µl

10 µM 2nd-SU-NCR2

1 µl

100 nM 2nd-SU-NR1

1 µl

1st PCR product1-5 µl*Sterile water

X µl

Total

50 µl

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Composition for C-terminal tag of 2nd PCR

KOD-Plus-Neo ver.
Reagents
10 X PCR buffer

5 µl

2 mM dNTPs

5 µl

25 mM MgSO4

3 µl

10 µM 2nd-T7-CF

1 µl

100 nM 2nd-protein tag-CR1

1 µl

10 µM 2nd-SU-NCR2

1 µl

1st PCR product1-5 µl*KOD

1 µl

Sterile water

X µl

Total

50 µl

PrimeSTAR ver.
Reagents
5 X PrimeSTAR buffer

10 µl

2.5 mM dNTPs

4 µl

10 µM 2nd-T7-CF

1 µl

100 nM 2nd-protein tag-CR1

1 µl

10 µM 2nd-SU-NCR2

1 µl

1st PCR product1-5 µl*Polymerase (2.5 U/µl)

0.5 µlSterile water

X µl

Total

50 µl

PrimeSTAR Max ver.
Reagents

2 X PrimeSTAR Max Premix

25 µl

10 µM 2nd-T7-CF

1 µl

100 nM 2nd-protein tag-CR1

1 µl

10 µM 2nd-SU-NCR2

1 µl

1st PCR product1-5 µl*Sterile water

X µl

Total

50 µl

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TRANSCRIPTION
Preparation of Transcription Reaction Solution
Prepare the transcription reaction solution on ice in a 1.5 ml tube as follows.
Please be careful not to mix 2nd PCR Product directly with 10x Transcription Buffer

Reagents (one reaction)
10 X Transcription buffer (TB)

2.5 µl

25 mM NTPs

2.5 µl

0.1 M DTT

1.25 µl

T7 RNA Polymerase

1 µl

2nd PCR product2.5 µlRNase-free water (DEPC)

15.25 µl

Total

25 µl

Transcription Reaction
1.

Tap the prepared transcription reaction solution lightly and use tabletop centrifuge to drop thesolution that attached to the wall gently.

2.

Close the cap securely to prevent evaporation of the solution.

3.

Incubate in water bath or incubator at 37 °C for 3 hours.

*The standard for reaction time is 3 hours up to 6 hours at most.
Transcription Purification
1.

After completion of the transcription reaction, load 1 μl transcription sample to agarose gelelectrophoresis.

2.

Add 10 μl of 4 M ammonium acetate per one sample (25 μl), mix well, add 100 μl of 100%
ethanol, inverted the tube, then use tabletop centrifuge for several seconds. Let stand for 20
minutes on ice.
Ammonium acetate is essential as neutralizing reagent. Please note that mRNA is inactivatedand proteins cannot be synthesized when purified with reagents for purification, trizole,ammonium sulfate, etc.

3.

Centrifuge at 15,000 rpm for 20 minutes at 4 °C

4.

Discard the supernatant with a pipete and use tabletop centrifuge for several seconds. Discard
the supernatant again by careful aspiration with a pipette. At this time, be careful to not touch
the pellet of the sample.

5.

Leave the pellet until dries. Keep the lid of the tube open, please be careful not to contaminate
impurities, dust, brushes.

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＊Nucleic acid dryer is not allowed
6. Add 70 μl of RNase-free water (DEPC water) per one sample (25 μl of transcription reaction
solution) to dissolve the mRNA pellet.
Suspend the pellet well and use it as an mRNA solution* Be careful as mRNA may adhere to the wall of the tube in a difficult state to check

Confirmation of Transcription
１．

Confirmation of transcription (mRNA)
After completion of purification, confirm the mRNA by electrophoresis.

２． Concentration measurement of transcription.
When measuring the concentration, purify phenol / chloroform system separately from thesample for translation.
Yield of one target mRNA from the transcription reaction is > 25 ug.

Depending on the target protein, mRNA may be synthesized in large quantities in thetranscription reaction, but when mRNA is subjected to the translation reaction in a large
amount (40 ug or more), on the contrary, the protein yield drops extremely, or the fulllength protein can not be obtained. Therefore, We suggest you can measure theconcentration, and in the case of 110 μl of translation solution in the next chapter, as
mRNA solution. Please prepare and supply to 25 ~ 40 ug / 70 ul, preferably 30 ~ 35 ug /70 ul of mRNA for translation per 110ul sample size.

In the case of coexpression using heterologous mRNA such as heterodimer, forexample, coexpression of protein with molecular weight A and molecular weight B,making the ratio of each mRNAs is A: B respectively and total amount of mRNA
about 30 to 35 ug / 70 ul, improves multimer of them.

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TRANSLATION
Preparation of translation reaction solution

Prepare the translation reaction solution with 1.5 ml tube or PCR tube as follows.
Prepare reagents other than mRNA first and make it to room temperature, then added to the mRNA
solution and resuspend gently so as not formed foam.
When mRNA concentration is unknown, it is suggested that whole mRNA synthesized by one
transcription reaction is added to one translation reaction solution.

Reagents
Wheat germ extract

20 µl

Amino acid mix

20 µl

mRNA

70 µl

Total

110 µlTranslation reaction (simple batch method)1.

Incubate the tube at 16 °C and let it react overnight (over 10 hours)

2.

Gently mix the sample by pipete, collected in an Eppendorf tube, centrifuged at 15,000 rpm
for 10 minutes at 4 ° C. The supernatant is stored at -80 ° C

Scale-Up
In this protocol, 1 translation reaction is 110 µl, but scale-up is possible.
For scaleup, please increase the ratio of translation reaction system as it is.

e.g.) When translation reaction 110 µl is scaled up to 220 µl, the composition is
Wheat germ extract
20ul -> 40ul
Amino acid mix
20ul -> 40ul
mRNA
70ul -> 140ul
If you scaled up, the yield of the target protein may be improved.
e.g.) In the case one translation reaction of 110 µl have a yield 10 µg, one translation reaction of
550 µl improves the yield per reaction solution with the target yield, about 70 µg

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Translation reaction (dialysis method)
Dialysis method can be used instead of simple batch method. In the dialysis method, insert thetranslation solution into the inside of the dialysis cup (sample solution), and use additional Amino acidmix as the dialysis external solution. Dialysis external solution is generally 20 times to 100 times thecapacity.
When using Slide-A-Lyzer ™ Mini dialysis tool (sample 0.1 mℓ, molecular weight cut off 3.5 K)
manufactured by Thermo Fisher Scientific Inc.

1. Place 2 ml of Amino Acid Mix into 1 well of a 24-well titer plate, diluted 4 times with ultrapure
water into, and cover the plate.＊ Since 2 ml Eppendorf tube matches the dialysis cup, thedialysis method can be do with 2 ml Eppendorf tube.
2.

Squeeze the dialysis cup into the lid of the plate.

3.

Take the total volume of 110 μℓ of the prepared translation reaction solution and transfer todialysis cup gently so bubbles do not enter on to the dialysis cup, two-step pushing of thepipette can causes foaming. Then close the lid of the dialysis cup. At this time, please set thelevel of the sample solution (reaction layer) and the level of the external dialysate (Amino Acid
Mix) are at the same level or the level of the sample solution is slightly lower.

4.

Incubate at 16 °C and let it react overnight (over 10 hours)

Moreover, by drilling 9mm hole in 24-well titer plate can fit to dialysis cup, but if it is difficult to drill,
use a floater to microtube.

 Since the volume of amino acid mix in the kit is not enough for dialysis method, please purchasethe optional Amino acid mix (product name: AAMX004) for dialysis method 4 reaction when
dialysis method is used.

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Confirmation of Protein

Confirm the translation product by acrylamide gel electrophoresis as follows.
1.

After completion of the translation reaction, fill up the translation reaction solution withultrapure water up to 3 volumes and stir gently.

2.

Centrifuge at 15,000 rpm for 20 min at 4 °C, collect supernatant.

3.

Load about 1-2 ul of sample to acrylamide gel, then electrophorese

4.

Detect according the staining method.

About the above procedure
Procedure 1. , The purpose of fill up is to save the detection sample, to avoid overloading, and tosolubilize the nonsolubilized compound. If it is Fill up up to three times, the buffer capacity of thereaction solution is maintained, but be careful when filling beyond this. If you do not need it, skip thisstep.
Procedure 2. Regarding precipitates, in addition to components derived from Wheat Germ, non solubilized objects may also be included. If necessary, resuspend the precipitate with an appropriate
Buffer to recover the desired product. If there is any doubt about the amount of target productrecovered from the supernatant, this will grasp the solubilization rate and improvisation by
optimizing the translation reaction solution.
Procedure 3. For electrophoresis samples, Western blot, silver staining, CBB staining, both aredetected at approximately 1-2 ul. In detection other than immunostaining, Wheat Germ components
are also detected, so loading large amounts of samples is not recommended.
The following is the result of synthesizing FLAG® fusion protein using this kit and detecting these
proteins by western blot using anti-FLAG® antibody.

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Cell-free Protein Synthesis Reagent Kit (PSS 3050) and Translation Reagent Set (TLS4050) are for research use only
KOD® and KOD-Plus-Neo are registered trademarks or trademarks of Toyobo Co., Ltd.
PrimeSTAR® and PrimeSTAR® MAX are registered trademarks or trademarks of Takara
Bio Inc.

FLAG® is a registered trademark of Sigma-Aldrich Co. LLC. Slide-A-Lyzer ™ is atrademark of Thermo Fisher Scientific Inc.
Other company names and product names mentioned may be other trademarks andregistered trademarks of each company.
The reagents, products, etc., are not always indicated by trademark indication ((R), TM).

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