User:University of Washington/6 August 2008

LuxR from AraC and TetR(Faifan)
-Tranformation of ligation product at room temp(P1010 on pSB1AC3 and promoter) succeed? There were too much growth, so the culture was streaked out in new Amp plate.

-Continued ligation process (the one at 4 degree Celcius overnight): denatured enzyme, filterd, tranformed into XL1-Blue, and grew on Amp plate.

-Discovered today that the dsDNA added for previous QuikChange reaction was way excessive due to unit misunderstanding.

-Did first part of QuikChange (set up reaction + theymocycling overnight)
 * nanodropped R0080(AraC) = 113.5 ng/ul, 1.9-260/280, 2.19-260/230
 * Elongation step 68 degree Celcius for 7 mins was removed in theymocycling.

MG1655Z1(Faifan)
-Got growth in Amp plate from MG1655Z1 stock. Have to find out what plasmid contains Amp resistance from literature.

-Got growth in Amp and a bit in Tet plates from MG1655Z1 Tsy#2 plate. This said that there were still some unknown plasmids in the strain. Two colonies from section 3 and 5 were picked and grew overnight in Tsy.

LuxR from pLac
-Glycerol stock of part I0462 made.

-Second R+E construction miniprepped and sent in for sequencing.

-Six aliquots of electrocompetent DH5a+LacIq cells made.

Lambda Red Recombineering of RP4 (Bryan)
Yesterday's recombinant cultures did not grow, indicating that recombination failed. However, the control culture grew overnight, indicating that the target plasmid is at least transforming the host cells.

The large size of RP1 may cause unique problems for recombineering. For example, a very large plasmid will have fewer molecules than a small plasmid for a given mass of plasmid DNA, perhaps affecting recombineering efficiency. Future troubleshooting will focus on optimizing the mass quantity of plasmid included in electroporation.

AHL Expression in Yeast (Bryan)
Gel-extracted restriction fragments of C0161 and yeast expression plasmids with Qiagen kit. Nanodrop indicated very low yield. Will re-attempt with "freeze & squeeze" extraction.

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