< Haynes Lab:Notebook | TB Biosensor | 2012 | 11
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Got colonies on 3/4 plates (Keio pSB1A3-Lacp-crtEIB, Keio pSB1A3-Lacp-LuxS, DH5alphaT pSB1A3-Lacp-LuxS). Ran colony PCR on 4 colonies from each positive plate.
Set up another Gibson assembly of the linear fusion proteins, crtE-PLflex-crtI and crtE-PLflex-crtB. Took 0.4ul from each and did PCR with the promoter-adding primers and 0.4ul from each for control PCR with P001 and P002, which will amplify just unassembled (or assembled) crtE. Then we purified the rest of the Gibson assembly using the Zymo Clean and Concentrator kit and eluted in 6ul. We did the same PCR as before using 1ul of the sample. I put the rest in the freezer so if the PCR works, we can check again to see if the purified samples are stable at -20. (note: according to NEB, T5 exonuclease cannot be heat inactivated).
Redid adding Gibson ends to PLFlex for fusion proteins because the previous batch had really low yields.