Week8 8/1/10-8/7/10

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Revision as of 21:25, 16 October 2010

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.

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 _____ --> plan to test competency