User:University of Washington/13 August 2008

From 2008.igem.org

(Difference between revisions)

Latest revision as of 22:34, 28 August 2008

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

@@ Line 4: / Line 4: @@
 <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>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>
@@ Line 18: / Line 18: @@
 - 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
 -Restriction Digest of BBa_Elowitz, BBa_E0240(GFP), BBa_I0462(LuxR), BBa_P1010(on pSB3K3)
-<table>
+*mix
-<tr><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>X(ul)</th><th>S(ul)</th><th>P(ul)</th></tr>
+<table border=1>
-<tr><th>BBa_Elowitz</th><th>32.5</th><th>EcoRI, 5</th><th>0.5</th><th>10</th><th>1</th><th>-</th><th>1</th><th>-</th></tr>
+<tr align="center">
-<tr><th>LuxR</th><th>32.5</th><th>NEB3, 5</th><th>0.5</th><th>10</th><th>-</th><th>1</th><th>-</th><th>1</th></tr>
+<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><th>GFP</th><th>32.5</th><th>NEB3, 5</th><th>0.5</th><th>10</th><th>-</th><th>1</th><th>-</th><th>1</th></tr>
+</tr>
-<tr><th>P1010</th><th>32.5</th><th>EcoRI, 5</th><th>0.5</th><th>10</th><th>1</th><th>-</th><th>-</th><th>1</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]]