Toehold Protocols
Dilution of DNA Fragments
Reagent | Volume | Final Concentration |
---|
Toehold Stock | 0.83 uL | - |
Nuclease Free Water | 5.17 uL | - |
- Pipette toehold stock and nuclease free water into a microcentrifuge tube
- Briefly vortex and spin dilution down
PCR
Reagent | Volume | Final Concentration |
---|
2X PhusionTM Plus PCR Master Mix | 25 uL | 1X |
Forward Primer | 2.5 uL | 0.5 uM |
Backward Primer | 2.5 uL | 0.5 uM |
5X PhusionTM GC Enhancer | 10 uL | 1X |
Template DNA | 1 uL | 5-100 ng genomic DNA |
Nuclease Free Water | 9 uL | - |
TOTAL | 50 uL | - |
1. Added reagents to a PCR tube according to the reaction chart above.
- One tube for GFP Toehold
- One tube for GFP Trigger
2. Ran tubes in Thermo Cycler for 1 hour using temperatures below:
- Initial Denaturation: 98°C for 30 sec
- Denaturation: 98°C for 10 sec
- Annealing: 61°C for 10 sec
- Extension: 72°C for 30 sec
- Cycle between denaturation, annealing, and extension for 30 cycles
- Final extension: 72°C for 5 min
- Infinite hold
PCR Cleanup
Reagent | Volume | Final Concentration |
---|
Buffer PB | 5 uL | - |
Sodium acetate | 10 uL | 3 M |
Buffer PE | 750 uL | - |
Nuclease Free Water | 30 uL | - |
- Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix.
- If mixture is orange or violet, add 10 uL of 3 M sodium acetate
- Place a QIAquick column in a 2 mL tube.
- Place buffer/DNA mixture into the QIAquick column and centrifuge for 60 sec at max speed. Disregard flow-through.
- Add 750 uL of Buffer PE to the QIAquick column and centrifuge for 60 sec at max speed. Disregard flow-through.
- Centrifuge the column once more to remove residual wash buffer.
- Place the column into a new clean microcentrifuge tube.
- Add 30 uL of nuclease free water to the column and let stand for 1 minute and then centrifuge.
myTXTL Pro Cell-Free Expression Kit
Reagent | Volume | Final Concentration |
---|
Pro Master Mix | 9 uL | - |
Pro Helper Plasmid | .5 uL | - |
Template DNA | 2.5 uL | X nm |
- Preheat the plate reader to 27°C
- Thaw all kit components and templates at room temperature, then immediately transfer to ice.
- Directly before use, spin down the myTXTL Pro Master Mix for 1 second with a mini-centrifuge followed by mixing the master mix with a pipette.
- Add all reagents into a 2mL Eppendorf tube on ice according to the table above.
- Briefly vortex and mini-centrifuge the assembled myTXTL reaction.
- Load the pre-warmed plate in the plate reader and start kinetics. On a (Plate reader suggested by paper: Biotek H1m) plate reader, the excitation is fixed at 485 nm, the emission at 525 nm, the time lapse between reads is typically 3 min for 16 h.
- Repeat the entire protocol as necessary for replicates.
Positive TXTL Reaction
Reagent | Volume | Final Concentration |
---|
Pro Master Mix | 9 uL | - |
Pro Helper Plasmid | .5 uL | - |
T7 deGFP Control Plasmid | 2.5 uL | - |
- Preheat the plate reader to 27°C
- Thaw all kit components and templates at room temperature, then immediately transfer to ice.
- Directly before use, spin down the myTXTL Pro Master Mix for 1 second with a mini-centrifuge followed by mixing the master mix with a pipette.
- Add all reagents into a 2mL Eppendorf tube on ice according to the table above.
- Briefly vortex and mini-centrifuge the assembled myTXTL reaction.
- Load the pre-warmed plate in the plate reader and start kinetics. On a (Plate reader suggested by paper: Biotek H1m) plate reader, the excitation is fixed at 485 nm, the emission at 525 nm, the time lapse between reads is typically 3 min for 16 h.
- Repeat the entire protocol as necessary for replicates.
Toehold Reactions (USING myTXTL Pro Cell-Free Expression Kit)
Reagent | Volume | Final Volume and Concentration |
---|
T7 Master Mix | 9 uL | - |
Pro Helper Plasmid | .5 uL | - |
Chi6 | 0.5 uL | 2 uM |
Toehold construct | 0.5 uL | 1 nM |
Trigger construct | 1.5 uL | 5 nM |
Nuclease Free Water | - | - |
TOTAL | 12 uL | - |
- Preheat the plate reader to 27°C
- Thaw all kit components and templates at room temperature, then immediately transfer to ice.
- Directly before use, spin down the myTXTL Pro Master Mix for 1 second with a mini-centrifuge followed by mixing the master mix with a pipette.
- Add all reagents into a 2mL Eppendorf tube on ice according to the table above.
- Briefly vortex and mini-centrifuge the assembled myTXTL reaction.
- Load the pre-warmed plate in the plate reader and start kinetics. On a (Plate reader suggested by paper: Biotek H1m) plate reader, the excitation is fixed at 485 nm, the emission at 525 nm, the time lapse between reads is typically 3 min for 16 h.
- Repeat the entire protocol as necessary for replicates.