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From 2010.igem.org

Contents 1 8/2/10 2 8/3/10 3 8/4/10 4 8/5/10 5 8/6/10

8/2/10

Competency test (iGEM T10 from Leonard T10) -->

cP 3A Assembly troubleshooting: why are there white colonies? --> Ran Gel along with Red and Green (lacp-gfp in cpc) (Sequencing necessary?)

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 **TRY PHOSPHATASE IN PARALLEL

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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.

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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 ________

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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)

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8/6/10

Lysed and stained (pMal-CHS3) cells with calcofluor white --> for chitin

Transformed ChiA knockout competent cells with _____ --> plan to test competency

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