User:University of Washington/13 August 2008

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== Conjugation ==
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== Conjugation (Scott) ==
experiment from [https://2008.igem.org/User:University_of_Washington/7_August_2008 Aug. 7] results<br>
experiment from [https://2008.igem.org/User:University_of_Washington/7_August_2008 Aug. 7] results<br>
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<table><
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<table border="1">
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<tr><th>experiment #</th><th>Bacteria</th><th>Yeast</th><th>Yeast count</th><th>transconjugation efficiency</th></tr>
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<tr><td>1</td><td>250uL</td><td>250uL</td><td>[[:Image:Conjugation 1.jpg| 66]]</td><td>2.64E-5</td></tr>
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<tr><td>2</td><td>200uL</td><td>300uL</td><td>[[:Image:Conjugation 1.jpg| 64]]</td><td>2.13E-5</td></tr>
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<tr><td>3</td><td>100uL</td><td>400uL</td><td>[[:Image:Conjugation 1.jpg| 32]]</td><td>8.00e-6</td></tr>
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<tr><td>4</td><td>50uL</td><td>450uL</td><td>[[:Image:Conjugation 1.jpg| 18]]</td><td>4.00e-6</td></tr>
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</table>
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==LuxR from AraC and TetR(Faifan)==
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<h4>QuikChange Approach</h4>
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- Overnight QuikChange#4 culture from Amp plate (reaction1 x 4, reaction2 x 2) in Tsy+Amp
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- Sequence of DNA from QuikChange#3 came back ==> No mutation.
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<h4>Elowitz</h4>
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-Made Glycerol Stock of BBa_Elowitz x 2
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-Restriction Digest of BBa_Elowitz, BBa_E0240(GFP), BBa_I0462(LuxR), BBa_P1010(on pSB3K3)
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*mix
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<table border=1>
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<tr align="center">
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<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>
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</tr>
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<tr align="center">
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<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>
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</tr>
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<tr align="center">
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<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>
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</tr>
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<tr align="center">
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<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>
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</tr>
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<tr align="center">
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<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>
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</tr>
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</table>
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*incubate 37 degree Celsius, 1.5 hours
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*15 mins water bath 80 degree Celsius
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*nanodrop at 230 nm
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<table border=1>
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<tr align="center">
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<th></th><th>ng/ul</th><th>260/280</th><th>260/230</th>
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</tr>
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<tr align="center">
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<td>BBa_Elowitz</td><td>76.2</td><td>1.12</td><td>0.34</td>
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</tr>
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<tr align="center">
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<td>LuxR</td><td>43.5</td><td>0.98</td><td>0.52</td>
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</tr>
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<tr align="center">
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<td>GFP</td><td>40.7</td><td>1.00</td><td>0.46</td>
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</tr>
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<tr align="center">
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<td>P1010</td><td>50.8</td><td>0.78</td><td>0.17</td>
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</tr>
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</table>
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*store in 4 degree Celsius
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==Lambda Red Recombineering of RP4 (Bryan)==
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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).
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==AHL Expression in Yeast (Bryan)==
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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.
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----
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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 #BacteriaYeastYeast counttransconjugation efficiency
1250uL250uL 662.64E-5
2200uL300uL 642.13E-5
3100uL400uL 328.00e-6
450uL450uL 184.00e-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_Elowitz32.5EcoRI, 50.5101-1-
LuxR32.5NEB3, 50.510-1-1
GFP32.5NEB3, 50.510-1-1
P101032.5EcoRI, 50.5101--1
  • incubate 37 degree Celsius, 1.5 hours
  • 15 mins water bath 80 degree Celsius
  • nanodrop at 230 nm
ng/ul260/280260/230
BBa_Elowitz76.21.120.34
LuxR43.50.980.52
GFP40.71.000.46
P101050.80.780.17
  • 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