Gate Anneals

Room Temperature Annealing

1. Mix gate and output in a 1.2:1 ratio in a tube.
2. Cover the tube in foil, if you didn't use an opaque/black tube.
3. Leave the tube on a bench top overnight.

Annealing With Heatblock

1. Preheat the heat block to 95 °C, add water to the well you will be using.
2. Put a piece of styrofoam (covered with foil) on the heat block. Make sure the temperature is still at 95 °C, and doesn't jump
3. Mix gate and output in a 1.2:1 ratio in a tube, put it in the heat block.
4. Cover again with the styrofoam, set the heat block to RT.
5. 5. Wait until the block cools. It will probably not reach RT, but will be at around 30 °C after a few hours. Turn the block off completely and wait for a bit (1h).

 N.B. It may be possible to anneal using a lower maximum temperature (say, 80 °C), as long as it is higher than the melting temperature of the oligos.

 *Adapted from Qian L, Winfree E. Scaling up digital circuit computation with DNA displacement cascades. , Science. 2011 Jun 3;332(6034):1196-201.

Plate Reader Studies

Kinetic Studies

1. Prepare wells: 100 µL 1X TAE, 12.5 mM Mg2+ buffer; 10 nM 100 µL RNA-ROX (gate); 10 nM 100 µL gate:output complex; 10 nM 100 µL gate:output complex.
2. Measure fluorescence of all wells for 1 minute (5 seconds between measurements).
3. Add 1 µL of the buffer to the buffer and gate wells. Add 1 µl of 1 µM input S6 to the first gate:output well; add 1 µL of 1 µM input S1 to the second gate:output well. Note: Mix well by pipetting into the well, after adding input shake the plate by doing a sort of swirling motion in a plane. If possible, set the plate reader to do this for you for 3 seconds with a 1 second rest time.
4. Measure fluorescence of all wells for 10 minutes (5 seconds between measurements).

RNA Transfection

RNA Transfection