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.

Protocol:

  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.

Notes:

  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.