TNT® Quick Coupled Transcription/Translation Systems Technical Manual TM045

Revised 8/22

TM045

TECHNICAL MANUAL

TnT

Quick Coupled

Transcription/Translation

Systems

Instructions for Use of Products

L1170, L1171, L2080 and L2081

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

www.promega.com TM045 · Revised 8/22

All technical literature is available at: www.promega.com/protocols/

Visit the web site to verify that you are using the most current version of this Technical Manual.

E-mail Promega Technical Services if you have questions on use of this system: techserv@promega.com



Quick Coupled Transcription/

Translation Systems

1. Description ............................................................................................................................................2

2. Product Components and Storage Conditions ..............................................................................................5

3. General Considerations ............................................................................................................................6

3.A. Transcription/Translation Considerations ........................................................................................... 6

3.B. Creating a Ribonuclease-Free Environment ........................................................................................8

3.C. Handling of 

Quick Master Mix ..................................................................................................8

4. Translation Procedure .............................................................................................................................8

4.A. General Protocol for TT

Quick Coupled Transcription/Translation Reactions Using Plasmid DNA .............8

  ............................................................................10

5. Post-Translational Analysis ....................................................................................................................11

5.A. SDS-Polyacrylamide Gel Analysis of Translation Products ..................................................................11

5.B. Western Blot Analysis ................................................................................................................... 12

  ..........................................................................................................13

  ..........................................................................................13

  ...................................................................13

7. Troubleshooting ...................................................................................................................................14

8. References .......................................................................................................................................... 16

9. Appendix ............................................................................................................................................. 16

9.A. Composition of Buffers and Solutions ............................................................................................. 16

  ....................................................................................................17

  ..................................................................... 19

9.D. Related Products.......................................................................................................................... 20

10. Summary of Changes ............................................................................................................................21

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TM045 · Revised 8/22 www.promega.com

1. Description

The 



the transcription and translation of DNA sequences cloned in plasmid vectors containing a T7 or SP6 promoter by

providing a rabbit reticulocyte extract that contains the T7 or SP6 RNA polymerase for transcription along with all

necessary components for translation (1). This system can produce moderate amounts of recombinant proteins (up to

5 micrograms of protein per milliliter of reaction) within an hour. The protein yields are lower when compared to E. coli

systems; however, this system is advantageous for the production of larger, more complex proteins, and can achieve









Quick

Coupled System can be used with PCR templates containing T7 promoters. The synthesized proteins can be labeled





Quick System is a luciferase-



protein. Refer to Section 6 for details.

Synthesizing a protein of the correct size is a useful way to verify the gene product of a particular DNA sequence. The



The amount of protein synthesized increases proportionally with reaction volume. Small amounts of target protein can be





cell-free system also can be used for a variety of functional studies, such enzymatic analysis, protein interactions and



Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

www.promega.com TM045 · Revised 8/22

Figure 1. Comparison of the 



Quick Coupled

Transcription/Translation System protocols.

TNT

Coupled Reticulocyte

Lysate System

Rabbit

Reticulocyte Lysate.

Add T

Reaction Buffer.

Add TNT

RNA Polymerase.

Add Amino

Acid Mixture

Minus Methionine.

Add RNasin

Ribonuclease Inhibitor.

TNT

Quick Coupled

Transcription/Translation

System

TNT

Quick

Master Mix.

Add label of choice.

Add DNA template and

Nuclease-Free Water.

Incubate at 30

C for

60-90 minutes.

Separate translation

products by SDS-PAGE.

Detect

1537MB11_2A

4 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

TM045 · Revised 8/22 www.promega.com

Figure 2. Studying protein:protein interactions using the 

Systems. This schematic shows translation of one protein

with radioactive [



S]methionine in a 







protein produced in the 

reaction. The sepharose is washed to remove unbound protein, and the remaining bound

proteins are eluted and analyzed on a gel. This technique allows measurement of the protein:protein interactions for both





Gene 1

TNT

System

Protein 1

GST Gene 1

Purify on Affinity Column

Express in E. coli

GST Protein 2

Western

W-Wash

E-Eluate

M-Marker

Autoradiography

W E M W E M

2598MA03_9A

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www.promega.com TM045 · Revised 8/22

2. Product Components and Storage Conditions

PRODUCT SIZE CAT.#

TnT

T7 Quick Coupled Transcription/Translation System 40 reactions L1170

TnT

SP6 Quick Coupled Transcription/Translation System 40 reactions L2080



•  



•  

•  



•  

•  

•  

PRODUCT SIZE CAT.#

TnT

T7 Quick Coupled Transcription/Translation System, Trial Size 5 reactions L1171

TnT

SP6 Quick Coupled Transcription/Translation System, Trial Size 5 reactions L2081



•  

Quick Master Mix

•  

•  



•  

Storage and Stability:

(avoid prolonged





Note that the systems are shipped in foil packaging because the system is sensitive to carbon dioxide released from dry

ice. If storing the system in a freezer containing dry ice, keep system components sealed in foil packaging for best

results. Do not 

of activity. The expiration date for the 

Quick Master Mix is listed on the product vial. Do not freeze-thaw the Master

Mix more than two times.

6 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

TM045 · Revised 8/22 www.promega.com

3. General Considerations

3.A. Transcription/Translation Considerations

DNA Template Considerations

Start Codon. Check the sequence on the DNA template for the presence of additional upstream start codons. During





initiation to occur prior to the desired start codon and result in a shift in the reading frame or production of a larger

protein than expected.

5´ UTR. 



Kozak Sequence. 



Stop Codon.

of RNAs.

PolyA. DNA constructs containing a poly(A) sequence downstream of the gene of interest generate more protein. Poly(A)



Plasmid Considerations

• The 

Quick Coupled Systems are optimized for use with circular plasmid templates, such as p™



• 



does not necessarily increase the amount of protein produced.

• 

T7 Quick Coupled System. We do not recommend



• 





• 

PCR Enhancer in each 50µl reaction.

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

www.promega.com TM045 · Revised 8/22

3.A. Transcription/Translation Considerations (continued)

PCR Considerations

• PCR templates can be used with the 

T7 Quick Coupled System. We do not recommend using PCR templates



Note:

T7 Quick for PCR



• 

optimal expression is often less than for inserts cloned into plasmid vectors (e.g., for a 500bp PCR product, use



Quick reaction).

• 

• 

PCR Enhancer in each 50µl reaction.

Primer Design Considerations for Incorporating a T7 Promoter into a PCR Product for in vitro Transcription/Translation.

Primer Required Desired

Forward Primer:

5´-(N

6–10







17–22



T7 promoter sequence













6–10 bases upstream of

promoter. Improves





17–22

)



promoter sequence and



transcription starts a few

bases upstream of the



better ribosome binding to

RNA.

Reverse Primer:



17–22





Needed to allow priming

of the target gene.

Reverse complement of

stop codon (TTA, CTA or

TCA). Terminates

translation.

Reverse complement of



8 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

TM045 · Revised 8/22 www.promega.com

3.B. Creating a Ribonuclease-Free Environment

To reduce the chance of RNase contamination, gloves should be worn when setting up experiments, and microcentrifuge

tubes and pipette tips should be RNase-free. It is not necessary to add Recombinant RNasin

Ribonuclease Inhibitor to the



Quick reactions to prevent degradation of RNA, because it is already present in the 

Quick Master Mix.

3.C. Handling of 

Quick Master Mix

Except for the actual transcription/translation incubation, all handling of the 

Quick Master Mix should be done at





possible after thawing to minimize loss of translational activity. Do not freeze-thaw the Master Mix more than two times.

4. Translation Procedure

The following is a general guideline for setting up a transcription/translation reaction. Also provided are examples of

standard reactions using [



S]methionine (radioactive), Transcend™ Non-Radioactive Detection System (colorimetric or







residues are incorporated into nascent proteins during translation. This biotinylated lysine is added to the transcription/











Note:www.promega.com/protocols/

4.A. General Protocol for TT

Quick Coupled Transcription/Translation Reactions Using Plasmid DNA

Materials to Be Supplied by the User

• 

• optional:or

• optional: or

• optional: 



or

• optional: Radiolabeled amino acid (for radioactive detection; see Section 5). We recommend using a grade of [











occur using other grades of label.





S]methionine (1,000Ci/mmol at 10mCi/ml) can be added to the TT

Quick





Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

www.promega.com TM045 · Revised 8/22

 

Quick Master Mix by hand-warming and place

on ice. The other components can be thawed at room temperature and then stored on ice.

 



to the bottom of the tube.

 

of any background incorporation of labeled amino acids.

 

5. Analyze the results of translation. See Section 5.A.

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TM045 · Revised 8/22 www.promega.com

 











tRNA.

See Section 6 for measurement of luciferase activity.

Table 1. Examples of 

Quick Reactions.



Components Unlabeled

Positive

Control [



S]Methionine

Transcend™

tRNA

FluoroTect™

Green



tRNA



Quick Master Mix

(a)

    



(b,c)

2µl

Methionine, 1mM 1µl 1µl – 1µl 1µl

[



S]methionine – – 2µl – –



(d)

– – 1–2µl –





tRNA

(d)

– – – 1–2µl



volume of

(e)

50µl 50µl 50µl 50µl 50µl

(a)

The 



have observed that certain gene constructs may differ in the Mg



concentrations required for optimal expression



magnesium acetate and potassium chloride are added to the 

Quick reaction. If using a construct with a viral leader,



PCR Enhancer.

(b)

Avoid adding calcium to the transcription/translation reaction. Calcium can reactivate the micrococcal nuclease used to

destroy endogenous RNA in the Master Mix and result in degradation of DNA or RNA templates.

(c)

We recommend using 1µl of the T7 

PCR Enhancer in a 50µl reaction to increase transcription/translation when

using PCR-generated DNA, linear plasmid or viral-enhanced plasmids.

(d)







detection of proteins that contain few lysines or are poorly expressed.

(e)

Small-scale reactions may be performed by reducing the recommended volumes proportionally.

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

www.promega.com TM045 · Revised 8/22

5. Post-Translational Analysis

Several methods can be used for detection of newly synthesized protein, depending on the labeling method chosen.

• 

Quick Master Mix contains roughly 100–200mg/ml



Coomassie



• Transcend™ biotinylated lysine-labeled proteins can be detected with streptavidin reagents on a Western blot. See

the Transcend™ Non-Radioactive Translation Detection System Technical Bulletin

• FluoroTect™

Green

Lys

in vitro Translation Labeling Systems Technical Bulletin

• 

5.A. SDS-Polyacrylamide Gel Analysis of Translation Products

The most widely applicable and versatile method for analysis of in vitro translation products is polyacrylamide gel

electrophoresis in the presence of 0.1% sodium dodecyl sulfate (SDS) and a discontinuous buffer system. Precast gels

are available from a number of manufacturers. In addition to convenience and safety, precast gels provide consistent

results.

Materials to Be Supplied by the User

• 



• 

• 

• 

 

 





Note: 

biotinylated tRNAs when used in the reaction. This step is optional.

 

 

Note:







www.promega.com/resources/pubhub/enotes/trouble-free-sdspage-analysis-of-proteins-synthesized-in-tnt-cell-free-

expression-systems/ .

12 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

TM045 · Revised 8/22 www.promega.com

 

too much of the reaction per lane can cause protein aggregation and poor separation of proteins.

6. Perform electrophoresis using standard conditions for your apparatus. Typically, electrophoresis is carried out at a

constant current of 20mA until the bromophenol blue dye has run to the bottom of the gel.

7. Detect target protein expression by one of the following methods:

 

 FluoroTect™ Green

Lys

vitro Translation Labeling Systems Technical Bulletin

c. Detection of biotinylated protein when labeling proteins with Transcend™ tRNA as directed in the Transcend™

Non-Radioactive Translation Detection Systems Technical Bulletin

5.B. Western Blot Analysis

Materials to Be Supplied by the User

• 

• Western blot apparatus

• 

• 

20)

• primary antibody for your protein of interest

• secondary antibody (against species of primary Ab)

• detection reagents (for label on secondary Ab)

• optional: 

 

 

 

 

20). Incubate for 1 hour

with gentle shaking.

 

Note: We recommend that you titrate your primary antibody dilutions to determine what dilution produces the best

results for your protein.

 

7. Incubate the membrane with the primary antibody at room temperature for 1 hour with gentle shaking.

 

shaking.

 

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

www.promega.com TM045 · Revised 8/22

 

 

12. Incubate the membrane with the secondary antibody for 1 hour with gentle shaking.

 



 

 

Materials to Be Supplied by the User

• luminometer

• 

• optional: 

• optional:or



 

 

2. Program the luminometer to perform a 2-second measurement delay followed by a 10-second measurement read

for luciferase activity.

 



 

 



manual. The reagent should be equilibrated to room temperature and mixed thoroughly by vortexing before beginning



 



 

 

14 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

TM045 · Revised 8/22 www.promega.com

7. Troubleshooting



available at: www.promega.com. E-mail: [email protected]

Symptoms Causes and Comments

 

used after more than two freeze-thaw cycles. Do not use reagents

after the expiration date. Prolonged exposure of unfoiled lysate to

dry ice can cause loss of activity.

Ethanol or salt present in the reaction may inhibit translation.

 





PCR Enhancer.

Calcium is present in the translation reaction. Avoid adding calcium

to the translation reaction. Calcium may reactivate the micrococcal

nuclease used to destroy endogenous mRNA in the lysate and result

in degradation of the DNA or mRNA template.

Ethanol present in the translation reaction. Residual ethanol

should be removed from template DNA preparations and amino

acids before they are added to the translation reaction.

Poly(A) tail. Addition of poly(A) tail to the template may increase

 

 



 

 

 



 

scanning for translation initiation can result in translation







[



S]methionine used is not translation grade or is beyond its



grades of [



S]methionine. We recommend Perkin-Elmer EasyTag™









Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

www.promega.com TM045 · Revised 8/22

Symptoms Causes and Comments

  

(continued) appears as a broad band migrating at 10–15kDa.

Aminoacyl tRNAs may produce background bands (~25kDa).

 



 



100mM DTT.

 

 





lysate and avoid this problem.

Smearing on the gel Too much protein loaded on the gel. Check the amount of sample

loaded on the gel and the amount of loading buffer. Too much

protein loaded can cause smearing.

Acrylamide concentration in the gel is too low. Acrylamide

concentration can be increased to 12%.

 

performing Western blots of the primary antibody.

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TM045 · Revised 8/22 www.promega.com

8. References

1. 

Eur. J. Biochem. 67

2. 

Biochim. Biophys. Acta. 1130

 Cell

62

 

Dependence on template structure. Nucl. Acids Res. 13

9. Appendix

9.A. Composition of Buffers and Solutions

T7 

PCR Enhancer

  

  

In nanopure water.

TBST

  

150mM NaCl

0.1% Tween

-20

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

www.promega.com TM045 · Revised 8/22

 



Quick Coupled Transcription/Translation

System. The Control DNAs contain the gene for luciferase under transcriptional control of a phage RNA polymerase





for use as control luciferase expression vectors only. They are not intended for use as cloning vectors.

ori

Luciferase

SP6

Control DNA

(4747bp)

Amp

luc

XmnI

(3651)

SP6 Initiation (1)

SP6 Promoter

HindIII (8)

NotI (21)

BamHI (41)

SacI

(1764)

XmnI

(1804)

(dA:dT)

1917VA04_6A

Additional description: Amp





SP6 RNA polymerase initiation 1

 

 

Poly(A) (dA)



 

 

 



 

 

 

18 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

TM045 · Revised 8/22 www.promega.com

 

ori

Luciferase

Control DNA

(4331bp)

Amp

XmnI

(2632)

luc

(dA:dT)

SacI (1767)

T7 Initiation (1)

T7 Promoter

I (44)

NotI (22)

HindIII (11)

1916VA04_6A

Additional description: Amp

 Sequence Reference Points:

T7 RNA polymerase initiation 1

 

 

Poly(A) (dA)



 



 

 

 

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

www.promega.com TM045 · Revised 8/22

 

Materials to Be Supplied by the User

• 



• 25% TCA/2% casamino acids

• 5% TCA

• 

• acetone

• 

• scintillation counter

 



 

 



 



 

 

10 minutes.

 





in a liquid scintillation counter.

 



 

proceed as described in Steps 1–7.

10. Perform the following calculation to determine percent incorporation:



× 100 = percent incorporation



11. Perform the following calculation to determine the fold stimulation over background:



= fold stimulation



20 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

TM045 · Revised 8/22 www.promega.com

9.D. Related Products





Product Size Cat.#



  

  



  



  



  



  



  

Translation Detection

Product Size Cat.#

  



Product Size Cat.#





  

Vectors

Product Size Cat.#

p

  

pCMV

  

  

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516

www.promega.com TM045 · Revised 8/22























10. Summary of Changes

The following changes were made to the 8/22 revision of this document:

1. Section 1 was updated.

 

 

 

5. The cover image and font were updated.