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User:University of Washington/11 August 2008
From 2008.igem.org
LuxR from AraC and TetR(Faifan)
-Miniprepped and sent sequence of ligated pSB1AC3 and promoter D29 (x3 of 25 degree Celsius ligation, x3 of 4 degree Celsius ligation)
-Grew overnight x3 from the single colony from QuikChange(trail#3) in Tsy + Amp
-Did Dpn1 reaction (continue from Ingrid's QuikChange#4), let sit from 3.5 hours, PCR purified and transformed into XL1-Blue (using 5ul DNA), grew on Amp plate.
Lambda Red Recombineering of RP4 (Bryan)
Troubleshooting of recombination continued. Attempted to optimize quantity of recombinant insert DNA. Performed two transformations, with 150 ng or 300 ng of insert. Neither culture grew O.N. Additionally, RP4 was already resident in the host, unlike previous experiments where both the target plasmid and insert were co-electroporated.
BLASTed Lambda Red primers against RP4's genome and confirmed that they are 100% homologous.
Is DY331 contaminated or is expression of Lambda Red genes not occurring?
Plan B is to use pKD78 as a Lambda Red helper plasmid.
AHL Expression in Yeast (Bryan)
Freeze n squeeze gel extraction of YEp and C0161 digest. Both yield and purity were somewhat improved over the Qiagen gel extraction kit, but still not ideal.
| Nanodrop Measurement | C0161 gel extract | GPD-YEp426 gel extract |
|---|---|---|
| 260/280 | 1.38 | 1.17 |
| 260/230 | 0.28 | 0.21 |
| [DNA](ng/ul) | 20.7 | 6.6 |
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