Took dialyzed bag with purified eGFP-RiAFP and divided it into 12 eppendorfs, containing 1 mL each. Flash-froze tubes in liquid nitrogen, then quickly used a hot edge to poke holes into top of eppendorfs to allow sublimation. Tubes then placed in lyophilizer for 3 nights.
Saturday:
Began cloning all pSB1AK8 constructs into pSB1C3 for parts submission (G3, H1, RiA, RiG, T2, Z2?) by digesting those 6 constructs and linearized pSB1C3 with EcoRI, PstI
Also, PCR amplified eGFP segment from original eGFP-RiAFP plasmid to use as a control for survival assays
Digested PCR fragment with eGFP with XbaI, PstI, and digested BBa_I712074 (strong T7 promoter) with SpeI and PstI
Gel extracted all digested pSB1AK8 constructs using kit, gel visualized with SYBRSafe (digestion seemed successful)
Sunday:
Ligated gel-extracted pSB1AK8 fragments with pSB1C3; ligated eGFP PCR fragment with BBa_I712074
Transformed ligations into DH5alpha cells, plated them
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