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User:University of Washington/31 July 2008
From 2008.igem.org
Contents |
LuxR from AraC and TetR
- minipreped BioBrick E0240
- ran gel for E0240: All 5 lanes had plasmids with same length.
- Transformed BBa_P1010 (contain death gene, cut from binder) into DB and XL1-Blue, then inoculated on Amp LB plate.
LuxR from pLac
-Gel ran on overnight digested DNA, again no DNA was found in gel for those lanes.
-Another restriction digest of parts R0010 and E0240 was started and allowed to incubate for 45 minutes. When gel was ran on these digests, DNA bands and spacing in gel was correct. E0240 insert and R0010 vector were isolated from gel and placed in the fridge.
Cure strain MG1655Z1
- minipreped Elowitz's plasmids
- ran gel for Elowitz's plasmid: Lane 2-5(colonies selected from plate of Glycerol Stock E.Coli) contained plasmids with same length. Lane 1(colony selected from original plate from Elowitz's lab) showed two bands, non of which matched with the rest of the lanes. Will further investigate the reasons tomorrow by restriction digestion of plasmid and compare.
- Selected 8 colonies of MG1655Z1 from Tsy plate and streaked out in new Tsy plate.
Lambda Red Recombination of RP4 (Bryan)
Nanodropped the following samples for optimization of transformation into Lambda Red recombinant strain DY331. Optimal qty. of recombinant vector DNA is 150 ng, optimal target plasmid qty. is 10 ng.
| Nanodrop Measurement | TetC/CmC Recombinant vector Sample 1 | TetC/CmC Recombinant vector Sample 2 | pUB307 (RP1 target plasmid) |
|---|---|---|---|
| 260/280 | 1.81 | 1.78 | 1.62 |
| 260/230 | 2.16 | 2.32 | 1.63 |
| [DNA](ng/ul) | 98.6 | 67.1 | 23.0 |
Rendered DY331 electrocompetent and immediately attempted four transformations. Trials 1 & 2 were at 1.8 V, resulting in time constants 3.2 & 3.0, respectively. Trials 3 & 4 were at 1.2 V, resulting in time constants 3.2 & 3.6. After outgrowth, trials 1 & 4 were plated on Cm media.
In case transformations fail, ON cultured more DY331 for tomorrow.
AHL Expression in Yeast (Bryan)
Nano-dropped yeast expression plasmids for optimization of digest & ligations.
| Nanodrop Measurement | GPD-YEp426 | AHD-YEp426 |
|---|---|---|
| 260/280 | 1.88 | 1.94 |
| 260/230 | 2.35 | 2.43 |
| [DNA](ng/ul) | 214.2 | 50.8 |
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