Toeholds

RNA-based riboregulators activated by complimentary triggers for gene quantification

Toehold Protocols

Dilution of DNA Fragments

ReagentVolumeFinal Concentration
Toehold Stock0.83 uL-
Nuclease Free Water5.17 uL-
  1. Pipette toehold stock and nuclease free water into a microcentrifuge tube
  2. Briefly vortex and spin dilution down

PCR

ReagentVolumeFinal Concentration
2X PhusionTM Plus PCR Master Mix25 uL1X
Forward Primer2.5 uL0.5 uM
Backward Primer2.5 uL0.5 uM
5X PhusionTM GC Enhancer10 uL1X
Template DNA1 uL5-100 ng genomic DNA
Nuclease Free Water9 uL-
TOTAL50 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:

  1. Initial Denaturation: 98°C for 30 sec
  2. Denaturation: 98°C for 10 sec
  3. Annealing: 61°C for 10 sec
  4. Extension: 72°C for 30 sec
  5. Cycle between denaturation, annealing, and extension for 30 cycles
  6. Final extension: 72°C for 5 min
  7. Infinite hold

PCR Cleanup

ReagentVolumeFinal Concentration
Buffer PB5 uL-
Sodium acetate10 uL3 M
Buffer PE750 uL-
Nuclease Free Water30 uL-
  1. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix.
    1. If mixture is orange or violet, add 10 uL of 3 M sodium acetate
  2. Place a QIAquick column in a 2 mL tube.
  3. Place buffer/DNA mixture into the QIAquick column and centrifuge for 60 sec at max speed. Disregard flow-through.
  4. Add 750 uL of Buffer PE to the QIAquick column and centrifuge for 60 sec at max speed. Disregard flow-through.
  5. Centrifuge the column once more to remove residual wash buffer.
  6. Place the column into a new clean microcentrifuge tube.
  7. 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

ReagentVolumeFinal Concentration
Pro Master Mix9 uL-
Pro Helper Plasmid.5 uL-
Template DNA2.5 uLX nm
  1. Preheat the plate reader to 27°C
  2. Thaw all kit components and templates at room temperature, then immediately transfer to ice.
  3. 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.
  4. Add all reagents into a 2mL Eppendorf tube on ice according to the table above.
  5. Briefly vortex and mini-centrifuge the assembled myTXTL reaction.
  6. 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.
  7. Repeat the entire protocol as necessary for replicates.

Positive TXTL Reaction

ReagentVolumeFinal Concentration
Pro Master Mix9 uL-
Pro Helper Plasmid.5 uL-
T7 deGFP Control Plasmid2.5 uL-
  1. Preheat the plate reader to 27°C
  2. Thaw all kit components and templates at room temperature, then immediately transfer to ice.
  3. 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.
  4. Add all reagents into a 2mL Eppendorf tube on ice according to the table above.
  5. Briefly vortex and mini-centrifuge the assembled myTXTL reaction.
  6. 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.
  7. Repeat the entire protocol as necessary for replicates.

Toehold Reactions (USING myTXTL Pro Cell-Free Expression Kit)

ReagentVolumeFinal Volume and Concentration
T7 Master Mix9 uL-
Pro Helper Plasmid.5 uL-
Chi60.5 uL2 uM
Toehold construct0.5 uL1 nM
Trigger construct1.5 uL5 nM
Nuclease Free Water--
TOTAL12 uL-
  1. Preheat the plate reader to 27°C
  2. Thaw all kit components and templates at room temperature, then immediately transfer to ice.
  3. 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.
  4. Add all reagents into a 2mL Eppendorf tube on ice according to the table above.
  5. Briefly vortex and mini-centrifuge the assembled myTXTL reaction.
  6. 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.
  7. Repeat the entire protocol as necessary for replicates.