User:University of Washington/13 August 2008

From 2008.igem.org

(Difference between revisions)
(LuxR from AraC and TetR(Faifan))
 
(3 intermediate revisions not shown)
Line 4: Line 4:
<table border="1">
<table border="1">
<tr><th>experiment #</th><th>Bacteria</th><th>Yeast</th><th>Yeast count</th><th>transconjugation efficiency</th></tr>
<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>66</td><td>2.64E-5</td></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>64</td><td>2.13E-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>32</td><td>8.00e-6</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>18</td><td>4.00e-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>
</table>
Line 18: Line 18:
- Sequence of DNA from QuikChange#3 came back ==> No mutation.  
- Sequence of DNA from QuikChange#3 came back ==> No mutation.  
-
<h4>Elowitz's</h4>
+
<h4>Elowitz</h4>
-Made Glycerol Stock of BBa_Elowitz x 2  
-Made Glycerol Stock of BBa_Elowitz x 2  
Line 44: Line 44:
*15 mins water bath 80 degree Celsius
*15 mins water bath 80 degree Celsius
*nanodrop at 230 nm
*nanodrop at 230 nm
-
<table>
+
<table border=1>
<tr align="center">
<tr align="center">
<th></th><th>ng/ul</th><th>260/280</th><th>260/230</th>
<th></th><th>ng/ul</th><th>260/280</th><th>260/230</th>
Line 62: Line 62:
</table>
</table>
*store in 4 degree Celsius
*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 #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