Rapid Bead-based Byte Assembly On-chip

What you will need:

• Annealed Anchor
• Annealed Terminator
• Prepared BioBytes
• Magnet
• Streptavidin-Coated Magnetic Microsphere "Beads" (4mg/mL)
• Wash/Binding Buffer - with no NaCl
• T4 DNA Ligase/Buffer
• USER™ (1 U/μL)
• Mineral Oil

Preparation:

1. Pipette 4.2 μL of each Byte into separate 1.5 mL microcentrifuge tubes (1 Byte per tube). As well, to each tube, also add 0.5 μL of ligase and 0.3 μL of ligase buffer.
2. The beads are stored at 4ºC, vortex briefly to resuspend the beads and dispense 2 μL of them into a tube. Add 3 μL of washing buffer. Return the stock of beads to 4ºC immediately.
3. Apply a magnet to the side of the tube, wait until solution clears completely, aspirate supernatant.
4. Wash beads by adding 5 μL of washing buffer and resuspend by flicking.
5. Apply the magnet as before, wait until solution clears completely, aspirate supernatant.
6. Repeat steps 4 & 5 once more.

On-chip protocol for construct creation and release

Remember: Add AB Bytes to BA Bytes and BA Bytes to AB Bytes. If you want a circular construct, include the Terminator as a Byte.
7. Fill the center chamber of the microfluidic chip with washing buffer. Observe the channels fill. If a channel is blocked, use another chip or use a pipette on the corresponding outer chamber to "suck" the washing buffer through from the center chamber.
8. Fill the central washing chamber to a higher level than the top of its chamber to induce Laplace flow.
9. Dispense 2 μL of beads into an empty chamber
10. Dispense 5 μL of the Anchor into a chamber.
11. Dispense 5 μL of each Byte (including the added ligase) into separate chambers. If any chambers have filled with washing buffer, pipette it out, then pipette the Byte in right away.
12. Using the magnet underneath the chip (either by hand, or with the robot), guide the beads out of their chamber, through the channel, through the washing chamber, and into the anchor chamber.
13. Mix every 2 minutes by moving the magnet side to side until 10 minutes is up.
14. Guide the beads to the washing chamber, then into a Byte chamber.
15. Mix every 2 minutes. After 15 minutes, guide the beads to the wash chamber, then to the next Byte chamber.
16. Repeat the previous step for the remaining Bytes.
17. After the last Byte is done, recover the beads by pipette into a 1.5 mL microcentrifuge tube. Wash the beads as in steps 4 to 5, resuspending the beads in 5 μL of dH20.
18. Add 1 μL 10x Pfu Buffer + 0.5 μL USER.
19. Incubate @ 37ºC 1 hr
20. Remove the beads by applying magnet, wait for solution to clear, aspirate supernatant into a fresh tube.
21. If circularizing: heat to 95ºC for 1 min, cool to RT slowly (let it sit on the bench).