ICA Preparation

Required Solutions:
- 2x BW Buffer
- 0.5x BW Buffer
- Elution Buffer
Prepared, working solutions of the Initator, Terminator and Capping Oligos
Purified, BsaI Digested MaSp Monomers
- Sufficient amount to complete ICA for the desired chain length.
- 50 ng monomer is used in each extension reaction.

Iterative Capped Assembly

Resuspend the M-270 Streptavidin beads by gently shaking the bottle.
Aliquot 5 uL of beads into tube. Apply the magnetic strip to isolate the beads.
- Wash twice with 100 uL of 2x BW Buffer
Resuspend the beads in 5 uL 2x BW Buffer, 4 uL ddH2O, and 1 uL 50 nM Initiator.
- Incubate on a rotator at RT for 45 minutes.
- This incubation period can be used to prepare the subsequent ligation reactions ahead of time. Mix all the relevant reagents together EXCEPT the T7 Ligase in separate tubes, one tube per reaction.
Apply the magnetic strip to isolate the beads. (All future wash steps require the magnetic strip to isolate the beads)
- Wash twice with 100 uL of 0.5x BW Buffer.
Add in the components of the first ligation:
- 5 uL 2x T7 Ligase Buffer
- 50 ng of piece AB
- ddH2O to 9.5 uL
Add 0.5 uL of T7 Ligase (temperature sensitive, put back in freezer between use)
Completely resuspend beads using pipet or by gently flicking.
Incubate on rotator at RT for 3 min.
Mix in components of second ligation:
- 5 uL 2x T7 Ligase Buffer
- 50 ng of piece BC
- 1 uL of 5 uM A-cap
- ddH2O to 9.5 uL
Add 0.5 uL T7 Ligase and resuspend completely.
Incubate on rotator at RT for 3 min.