Quick Start Protocol for NEBExpress® Cell-free E. coli Protein Synthesis System

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
    Control DHFR-His template (125 ng/μl) --- 2 μl  ---
    Plasmid template (>100 ng/μl)  --- --- 250 ng
    Water 11 μl 9 μl To 50 μl

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

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

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

Guidelines for troubleshooting can be found in this chart.