Glycerol Stock

What you will need:

• Overnight with bacterial growth
• Screw cap tubes
• Glycerol

Preparation:

1. Prepare X 1.5 microcentrifuge tubes, where X is the number of constructs to be made.
2. The beads are stored at 4ºC, vortex briefly to resuspend the beads and dispense 40 μL into each tube. Return the stock of beads to 4ºC immediately.
3. Apply the magnet, wait until solution clears completely, aspirate supernatant.
4. Wash beads by adding 75 μL of “Wash/Binding Buffer” and resuspend by flicking.
5. Apply the magnet, wait until solution clears completely, aspirate supernatant.
6. Repeat steps 4 & 5 once more.

• Pipet 150 uL 50% glycerol into 3* properly labeled 1.5 ml screw cap tubes.
• Add 350 uL of overnight culture to each tube.
• Pipet up and down to gently mix.
• Flash freeze in liquid N2 or dry ice/methanol bath
• Place in -80 C freezer when frozen.

Anchor

7. Apply 20 μL of Anchor (A or B) to the bead solution
8. Incubate at room temperature for 10 minutes
9. Apply the magnet, wait until solution clears completely, aspirate supernatant.
10. Wash twice as in steps 4-6.
11. Wash once more with 75 μL 1X ligase buffer.

BioByte Addition and Construct Release

12. Add 20 μL of the first Byte, resuspend beads, then add 2.3 μL 10x Ligase Buffer + 1 μL Ligase.
13 Incubate at room termpature for 20 minutes, every few minutes resuspend the beads by flicking the tube. BE GENTLE and tap the droplets back to the bottom of the tube after mixing.
14. Wash twice as in steps 4-6 with Wash Buffer and once more with 1X ligase buffer.
15. Repeat steps 12-14 for each BioByte to be added.
If you wish to circularize:
16. Add 20 μL of Terminator (A or B), resuspend beads, then add 2.3 μL 10x Ligase Buffer + 1 μL Ligase.
17. Incubate at room temperature for 20 minutes
18. Wash twice with Wash Buffer and once more with 1X ligase buffer.
Release from the bead:
19. Resuspend beads in 20 μL dH2O.
20. Add 2.5 μL 10x Pfu Buffer + 1 μL USER.
21. Incubate @ 37ºC 1 hr
22. Remove the beads by applying magnet, wait for solution to clear, aspirate supernatant into a fresh tube.
23. Heat to 95ºC for 1 min, cool to RT slowly (let it sit on the bench).

Transformation