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.
- Thaw all components on ice.
- Gently vortex the NEBExpress™ S30 Synthesis Extract and Protein Synthesis Buffer to mix.
- Combine reagents in a 1.5 ml microcentrifuge tube on ice as follows:
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
- Incubate reactions at 37°C, with shaking, for 2-4 hours.
- Analyze by method of choice or freeze at -20°C for later use.
- 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.
- Optimal GamS concentration must be determined empirically for a particular target.
- 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.