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User:University of Washington/13 August 2008
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| + | == Conjugation (Scott) == | ||
| + | |||
| + | experiment from [https://2008.igem.org/User:University_of_Washington/7_August_2008 Aug. 7] results<br> | ||
| + | <table border="1"> | ||
| + | <tr><th>experiment #</th><th>Bacteria</th><th>Yeast</th><th>Yeast count</th><th>transconjugation efficiency</th></tr> | ||
| + | <tr><td>1</td><td>250uL</td><td>250uL</td><td>[[:Image:Conjugation 1.jpg| 66]]</td><td>2.64E-5</td></tr> | ||
| + | <tr><td>2</td><td>200uL</td><td>300uL</td><td>[[:Image:Conjugation 1.jpg| 64]]</td><td>2.13E-5</td></tr> | ||
| + | <tr><td>3</td><td>100uL</td><td>400uL</td><td>[[:Image:Conjugation 1.jpg| 32]]</td><td>8.00e-6</td></tr> | ||
| + | <tr><td>4</td><td>50uL</td><td>450uL</td><td>[[:Image:Conjugation 1.jpg| 18]]</td><td>4.00e-6</td></tr> | ||
| + | </table> | ||
| + | |||
| + | ==LuxR from AraC and TetR(Faifan)== | ||
| + | |||
| + | <h4>QuikChange Approach</h4> | ||
| + | |||
| + | - Overnight QuikChange#4 culture from Amp plate (reaction1 x 4, reaction2 x 2) in Tsy+Amp | ||
| + | |||
| + | - Sequence of DNA from QuikChange#3 came back ==> No mutation. | ||
| + | |||
| + | <h4>Elowitz</h4> | ||
| + | |||
| + | -Made Glycerol Stock of BBa_Elowitz x 2 | ||
| + | |||
| + | -Restriction Digest of BBa_Elowitz, BBa_E0240(GFP), BBa_I0462(LuxR), BBa_P1010(on pSB3K3) | ||
| + | *mix | ||
| + | <table border=1> | ||
| + | <tr align="center"> | ||
| + | <th></th><th>dH2O(ul)</th><th>Buffer(ul)</th><th>BSA(ul)</th><th>DNA(ul)</th><th>E(ul)</th><th>X(ul)</th><th>S(ul)</th><th>P(ul)</th> | ||
| + | </tr> | ||
| + | <tr align="center"> | ||
| + | <td>BBa_Elowitz</td><td>32.5</td><td>EcoRI, 5</td><td>0.5</td><td>10</td><td>1</td><td>-</td><td>1</td><td>-</td> | ||
| + | </tr> | ||
| + | <tr align="center"> | ||
| + | <td>LuxR</td><td>32.5</td><td>NEB3, 5</td><td>0.5</td><td>10</td><td>-</td><td>1</td><td>-</td><td>1</td> | ||
| + | </tr> | ||
| + | <tr align="center"> | ||
| + | <td>GFP</td><td>32.5</td><td>NEB3, 5</td><td>0.5</td><td>10</td><td>-</td><td>1</td><td>-</td><td>1</td> | ||
| + | </tr> | ||
| + | <tr align="center"> | ||
| + | <td>P1010</td><td>32.5</td><td>EcoRI, 5</td><td>0.5</td><td>10</td><td>1</td><td>-</td><td>-</td><td>1</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | *incubate 37 degree Celsius, 1.5 hours | ||
| + | *15 mins water bath 80 degree Celsius | ||
| + | *nanodrop at 230 nm | ||
| + | <table border=1> | ||
| + | <tr align="center"> | ||
| + | <th></th><th>ng/ul</th><th>260/280</th><th>260/230</th> | ||
| + | </tr> | ||
| + | <tr align="center"> | ||
| + | <td>BBa_Elowitz</td><td>76.2</td><td>1.12</td><td>0.34</td> | ||
| + | </tr> | ||
| + | <tr align="center"> | ||
| + | <td>LuxR</td><td>43.5</td><td>0.98</td><td>0.52</td> | ||
| + | </tr> | ||
| + | <tr align="center"> | ||
| + | <td>GFP</td><td>40.7</td><td>1.00</td><td>0.46</td> | ||
| + | </tr> | ||
| + | <tr align="center"> | ||
| + | <td>P1010</td><td>50.8</td><td>0.78</td><td>0.17</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | *store in 4 degree Celsius | ||
| + | |||
| + | ==Lambda Red Recombineering of RP4 (Bryan)== | ||
| + | |||
| + | Attempted the following transformations RP1 + pKD78, RP4 + pKD78, and pKD78 alone. Selected with cam/tet, cam/tet, and cam, resepectively. None of the O.N. cultures grew. Possible problems are imprecision in micropipetting due to extremely small volumes <0.5 ul of vector DNA or not enough cells (1 mL of culture per electroporation). | ||
| + | |||
| + | ==AHL Expression in Yeast (Bryan)== | ||
| + | |||
| + | Transformed C0161/YEp426 ligation produce from yeseterday into XL-1 Blue. O.N. culture grew in amp, indicating successful transformation. Miniprepped and made glycerol stock. | ||
| + | |||
---- | ---- | ||
Back to [[Team:University_of_Washington/Notebook#Notebook]] | Back to [[Team:University_of_Washington/Notebook#Notebook]] | ||
Latest revision as of 22:34, 28 August 2008
Contents |
Conjugation (Scott)
experiment from Aug. 7 results
| experiment # | Bacteria | Yeast | Yeast count | transconjugation efficiency |
|---|---|---|---|---|
| 1 | 250uL | 250uL | 66 | 2.64E-5 |
| 2 | 200uL | 300uL | 64 | 2.13E-5 |
| 3 | 100uL | 400uL | 32 | 8.00e-6 |
| 4 | 50uL | 450uL | 18 | 4.00e-6 |
LuxR from AraC and TetR(Faifan)
QuikChange Approach
- Overnight QuikChange#4 culture from Amp plate (reaction1 x 4, reaction2 x 2) in Tsy+Amp
- Sequence of DNA from QuikChange#3 came back ==> No mutation.
Elowitz
-Made Glycerol Stock of BBa_Elowitz x 2
-Restriction Digest of BBa_Elowitz, BBa_E0240(GFP), BBa_I0462(LuxR), BBa_P1010(on pSB3K3)
- mix
| dH2O(ul) | Buffer(ul) | BSA(ul) | DNA(ul) | E(ul) | X(ul) | S(ul) | P(ul) | |
|---|---|---|---|---|---|---|---|---|
| BBa_Elowitz | 32.5 | EcoRI, 5 | 0.5 | 10 | 1 | - | 1 | - |
| LuxR | 32.5 | NEB3, 5 | 0.5 | 10 | - | 1 | - | 1 |
| GFP | 32.5 | NEB3, 5 | 0.5 | 10 | - | 1 | - | 1 |
| P1010 | 32.5 | EcoRI, 5 | 0.5 | 10 | 1 | - | - | 1 |
- incubate 37 degree Celsius, 1.5 hours
- 15 mins water bath 80 degree Celsius
- nanodrop at 230 nm
| ng/ul | 260/280 | 260/230 | |
|---|---|---|---|
| BBa_Elowitz | 76.2 | 1.12 | 0.34 |
| LuxR | 43.5 | 0.98 | 0.52 |
| GFP | 40.7 | 1.00 | 0.46 |
| P1010 | 50.8 | 0.78 | 0.17 |
- store in 4 degree Celsius
Lambda Red Recombineering of RP4 (Bryan)
Attempted the following transformations RP1 + pKD78, RP4 + pKD78, and pKD78 alone. Selected with cam/tet, cam/tet, and cam, resepectively. None of the O.N. cultures grew. Possible problems are imprecision in micropipetting due to extremely small volumes <0.5 ul of vector DNA or not enough cells (1 mL of culture per electroporation).
AHL Expression in Yeast (Bryan)
Transformed C0161/YEp426 ligation produce from yeseterday into XL-1 Blue. O.N. culture grew in amp, indicating successful transformation. Miniprepped and made glycerol stock.
Back to Team:University_of_Washington/Notebook#Notebook
