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From 2014.igem.org

We miniprepped pHBD 21 and 24.

We retransformed pHBD 5 and 35. pHBD 37 arc’ed so we decided to pick from the original biobrick plate tomorrow morning when we inoculate cultures for the others to miniprep.

We mini-prepped pHBD 36, 37, 38, and pHBD4

Ran the PCR from yesterday ((pBbE1a_Tet_Chromoprotein fragments and bb) on a gel. The resulting image was streaky and weird. I hypothesize it is because I used millipore water instead of TAE to make the gel (sorry).

The new gibsons of pHBD 21 and 24 plated from yesterday showed more colonies than the first attempt. Jose was still suspicious. We ran another diagnostic PCR to amplify the expected insert.

We ran the PCRs for pHBD39-44 now that we had the mini-prepped pHBD 36,37, 38 plasmids so we could PCR all chromoproteins (See PCR planning for details). We ran them all on a gel and everything worked except for the pHBD37 insert (yellow chromoprotein). We also ran the gibsons of pHBD21 and 24 on this gel (the 4 lanes next to the 100 bp ladder).

The gibsons of pHBD 21 & 24 were not exactly what we expected. Although confused, we continued to miniprep them (pHBD 21 & 24), so that we could sequence them and figure out what is going on. For the rest, we extracted the bands that we could and ran the gibsons for pHBD39-44. We then transformed the gibsons by electroporation into MACH1 cells and plated them.

We received primers to put hybB into the SB3K3 backbone. We ran the PCR and ran it on a gel:

(Insert gel image here)

The PCR for the backbone didn’t work. We decided to scrap the gel and troubleshoot the issue with the pSB3K3 backbone.

To do: sequence pHBD 21 & 24 from minis

Activity Log

Transformed F12, F24, F48 vectors into Mach I cells Mini- prepped Mach I cells with F12, 24, and 48 linkers Ran PCR on the plasmids