Iterative Capped Assembly (ICA) requires 50 ng of fragment for each extension cycle.
DNA Amplification
Starting from fully sequenced plasmids, DNA can be amplified in two ways.
PCR Amplification
Amplify 220 pg of each plasmid in a 50 uL reaction with VF2 and VR primers (the biobrick sequencing primers) and using Q5 polymerase and the GC enhancer. Set up 8x 50 uL reactions.
Anneal at 63 C, using 18 cycles.
Pool all 8 reactions together, and PCR purify
Maxiprep (Cloning Amplification)
Streak the glycerol stocks on chloramphenicol plates, and grow overnight at 37 C.
Pick one colony per construct into 100 mL liquid culture. Grow overnight at 37 C.
Harvest and prepare cells according to Qiagen Maxiprep protocol.
BsaI Digestion
Use 4 uL NEB BsaIin a 50 uL reaction to digest either:
1.0 to 1.5 ug of PCR amplified MaSp.
5 ug of whole plasmid.
Digest at 50 C for 2 hrs, then heat kill at 65 C for 20 min.
Run all of the digest mixture on 1.8% gel for purification.
Excise 102 bp band, and use Qiagen Kit to gel extract.
