"

From 2010.igem.org

You do not have permission to edit this page, for the following reason:

You can view and copy the source of this page:

__NOTOC__ ===Week8 Summary=== We tried our 3A assemblies using circular plasmid backbones extracted from the iGEM kit instead of the PCRed linear backbone plasmid. This solved our 3A assembly problem and all assemblies worked. We assembled our CP-LacPi inducible promoter parts. We also made competent ChiA knockout cells. We began assembling our (CP-LacPi)-(GFP) constructs to test/characterize the CP-LacPi parts. We assembled pMAL-CHS3 and tried to stain for chitin using calcofluor white. ---- ===8/2/10=== cP 3A Assembly troubleshooting: why are there white colonies? --> Ran Gel along with Red and Green (lacp-gfp in cpc) 1050 (rfp default insert), 2150 (bbp), 55+878 (lacp,gfp) red should be - 3200, 1050, 2150 white should be - 2150 green should be - 3083, 933, 2150 cP 3A Assembly troubleshooting: How to improve efficiency? Tried Phosphatase in parallel. ---- ===8/3/10=== Gel Results (White, Green, Red Colonies from 3A Assembly) *Red colonies look normal *Green colonies look normal *White colonies --> abnormal bands at 750bp, 1750bp, 2600bp, and 4300bp The white colonies could be due to aggregation of GFP or RFP protein due to excessive amounts of plasmid. Inoculated CP1-LacPi1, CP2-LacPi1, CP3-LacPi1, CP1-LacPi2, CP2-LacPi2, CP3-LacPi2, Holin1-XX, Holin2-XX, PMAL, GFP, LacPi1, LacPi2, YdgG Knockout, ChiA Knockout. ---- ===8/4/10=== Miniprepped CP1-LacPi1, CP2-LacPi1, CP3-LacPi1, CP1-LacPi2, CP2-LacPi2, CP3-LacPi2, Holin1-XX, Holin2-XX, PMAL, GFP, LacPi1, LacPi2. Miniprepped (CP1-Lacpi1) part --> digested with E/P --> ran a gel *Should be 35+1372/1370 = 1405/1407 +20(pre/suffix) Miniprepped (Holin1-XX) and (Holin2-XX) part --> digested with E/P ran a gel *Should be 1257, 317 + 129 = 1386, 446 +20 (prefix/suffix) Made glycerol stock of ________ ---- ===8/5/10=== Made competent ChiA knockout cells Made electrocompetent ECNR2 cells Minipreppred (pMal-CHS3) part Assembled and transformed (CP1-LacPI1)-(GFP), (CP2-LacPI1)-(GFP),(CP3-LacPI1)-(GFP),(CP1-LacPI2)-(GFP),(CP2-LacPI2)-(GFP), (CP3-LacPI2)-(GFP) ---- ===8/6/10=== Lysed and stained (pMal-CHS3) cells with calcofluor white --> for chitin Transformed ChiA knockout competent cells with pUC19 --> plan to test competency ----

Return to Week8 8/1/10-8/7/10.