Typical Protocol for NEBExpress® GamS Nuclease Inhibitor when used with the NEBExpress® Cell-free E. coli Protein Synthesis System (NEB #E5360)

Using a positive control template to verify protein synthesis can be useful when unfamiliar with in vitro transcription-translation protocols. To prevent nuclease contamination, wear gloves and use nuclease-free tubes and tips. Keep all reagents on ice before and during the assembly of reactions and avoid repeated freeze-thaw cycles of the tubes. Reactions are typically 50 μL but can be scaled down or up, as needed. Reactions are typically assembled in nuclease-free 1.5 mL microcentrifuge tubes. Components can be pre-assembled to create a master mix for a desired number of reactions. Use the master mix immediately, discard any unused master mix.


  1. Thaw all components on ice.

  2. Gently vortex the NEBExpress™ S30 Synthesis Extract and Protein Synthesis Buffer to mix.

  3. Combine reagents in a 1.5 ml microcentrifuge tube on ice as follows: 

     Negative Control
    (no DNA)
    Positive Control Sample
     NEBExpress™ S30 Synthesis Extract  12 μl  12 μl  12 μl
     Protein Synthesis Buffer (2X)  25 μl  25 μl  25 μl
     T7 RNA Polymerase  1 μl  1 μl  1 μl
    RNase Inhibitor, Murine  1 μl  1 μl  1 μl
     DHFR-His control template (125ng/µL)  ---  2 μl ---
     Linear DNA template (>100ng/µL)  --- --- 250 ng
     NEBExpress™ GamS Nuclease Inhibitor      1 μl
     Water  11 μl  9 μl  To 50 μ

  4. Incubate reactions at 37°C, with shaking, for 2-4 hours.

  5. Analyze by method of choice or freeze at -20°C for later use.


  1. During the experimental setup, it is recommended to add the linear DNA template in the last step to allow GamS to bind and inhibit RecBCD exonuclease before RecBCD has a chance to act on the DNA. 
  2. Optimal GamS concentration must be determined empirically for a particular target.
  3. Additional incubation time (maximum 10 hours) at 37°C may increase yield.

Guidelines for template design and preparation as well as tips for optimizing protein synthesis can be found in the E5360 product manual.