Team:Chiba/Calendar-Home/1 September 2008

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Revision as of 20:54, 29 October 2008

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31 August 2008 <|> 2 September 2008

Laboratory work

Team:Input

Ptet＋RBS＋cIの、Mini Prep産物の濃度チェック。
-Ptet-cI-pMB1-Amp-の機能チェック。
-Ptet-cI-pMB1-Amp-,-PcI-GFP-p15A-Cm-をDouble Transformation(BWΔflic)
結果-->GFPが発現していた。-->-PcI-GFP-p15A-Cm-が機能していないのでは？
ターミネーターをつける、pSB1A3にのせかえる。
-Ptet-cI-pMB1-Amp-のDigestion Check

混ぜ表

	Double	Single
dH₂O(μL)	1	2
XbaI(μL)	1	1
SpeI(μL)	1	-
BSA(×10)(μL)	1	1
NEB(×10)(μL)	1	1
DNA(μL)	5	5
TOTAL(μL)	10	10

UV irradiation test

two plasmids from ?
- -Ptrc-LuxR-Plux-cI-colE1-Amp-
- -PcI-GFP-p15a-Cm-の2plasmid(BW)

Incubated cultures(from Glycerol Stocks) with 2mL of LB-Ampicillin Medium for 12 hours at 37 degrees.
UV⊕:5.21,UV⊖:5.38<--?
moved the cultures to small plates and started UV-irradiation.(wavelength:254nm,distance from the UV lamp to the cultures were 7.5cm.UV⊖のものは暗所に置いておく。)
covered the plates with polyethylene wrap.
after irradiation(Sample:0min,10min,30min,1h,2h,4h,6h,8h)(Negative Control:0h,1h,4h,8h)

よく攪拌してから、20μl採取。

diluted each cultures with LB-ampicillin medium 10⁴-fold and 10⁵-fold (volume/volume).
incoculated 20μl of the cultures and incubated for 12 hours at 37 degrees.
counted the CFU(determined viable cell count).

Viable cell count

	UV+ 10⁴	UV+ 10⁵	UV-10⁴	UV-10⁵
0min	423	74	271	156
10min	301	51	-	-
30min	139	7	-	-
1h	89	5	1040	54
2h	5	1	-	-
4h	2	0	254	50
6h	0	0	-	-
8h	1	0	1155	106

Team:Communication

(31/8)--->Gel Check

Sample No.	1~3	4
Sample DNA	20	15
Loading Dye	4	3
TOTAL	24μl	18μl

From left;

insert-1([http://partsregistry.org/Part:BBa_I9026 BBa_I9026])

insert-2([http://partsregistry.org/Part:BBa_I9030 BBa_I9030])

insert-3([http://partsregistry.org/Part:BBa_S03154 BBa_S03154])

vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010])

--->Gel extract

--->zymo

insert-1([http://partsregistry.org/Part:BBa_I9026 BBa_I9026]) -> 7μL

insert-2([http://partsregistry.org/Part:BBa_I9030 BBa_I9030]) -> 7μL

insert-3([http://partsregistry.org/Part:BBa_S03154 BBa_S03154]) -> 7μL

vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010]) -> 15μL

--->SAP

vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010])

--->Zymo

vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010]) -> 20μL

--->Gel Check

Sample DNA	1
Loading Dye	1
dH₂O	4
TOTAL	6μl

From left;

insert-1([http://partsregistry.org/Part:BBa_I9026 BBa_I9026]) -> OK
insert-2([http://partsregistry.org/Part:BBa_I9030 BBa_I9030]) -> OK
insert-3([http://partsregistry.org/Part:BBa_S03154 BBa_S03154]) -> None --> Transformation [http://partsregistry.org/Part:BBa_S03154 BBa_S03154]-->

(2/9)Mini prepwith [http://partsregistry.org/Part:BBa_K084009 BBa_K084009], [http://partsregistry.org/Part:BBa_K084010 BBa_K084010]

vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010])

--->Ligation

Sample No.	(1)	(2)	(3)	(4)	(5)
insert-1([http://partsregistry.org/Part:BBa_I9026 BBa_I9026])	3	-	3	-	-
insert-2([http://partsregistry.org/Part:BBa_I9030 BBa_I9030])	-	3	-	3	-
vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010])	3	3	-	-	3
ligase	1	1	1	1	1
Buffer	1	1	1	1	1
dH₂O	2	2	5	5	5
TOTAL	10μl	10μl	10μl	10μl	10μl

--->Transformation

Competent cells : XL10GOLD 30μL

Transformed the following and grew on new ampicillin plates.

[http://partsregistry.org/Part:BBa_K084009 BBa_K084009(Plac+RBS+RhlI+LVA, Amp)] -> 628 colonies
[http://partsregistry.org/Part:BBa_K084010 BBa_K084010(Plac+RBS+CinI+LVA, Amp)] -> 500 colonies
insert-1(RBS+RhlI+LVA) -> 9 colonies
insert-2(RBS+CinI+LVA) -> No colonies on the plate
vector-4(Plac, Amp) -> 186 colonies

--->(2/9) Colony PCR

Transformation

Competent cells : JW1908 40μL

Transformed the following and grew on new ampicillin plates.

[http://partsregistry.org/Part:BBa_K084007 BBa_K084007(Plac+RBS+LasI)]
[http://partsregistry.org/Part:BBa_K084008 BBa_K084008(Plac+RBS+RhlI)]
[http://partsregistry.org/Part:BBa_K084010 BBa_T9002(Ptet+RBS+LuxR+GFP)]

--->(2/9)Liquid Culture

Team:Output

Ligation

vector:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010] insert:[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]①
negative control:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010]②

Sample No.	①	②
vector	2	2
insert	3	0
Ligase Buffer	2	2
Ligase	1	1
dH₂O	3	5
TOTAL	10μl	10μl

-->R/T 2hour

Transformation

[http://partsregistry.org/Part:BBa_R0079 BBa_R0079]
[http://partsregistry.org/Part:BBa_R0071 BBa_R0071]
[http://partsregistry.org/Part:BBa_R0077 BBa_R0077]
[http://partsregistry.org/Part:BBa_R0078 BBa_R0078]
[http://partsregistry.org/Part:BBa_R0062 BBa_R0062]

@@ Line 120: / Line 120: @@
 <tr>
 <td>TOTAL</td>
-<td>24</td><td>18</td>
+<td>24μl</td><td>18μl</td>
 </tr>
 </table>
 :From left;
-::insert-1(I9026)
+::insert-1([http://partsregistry.org/Part:BBa_I9026 BBa_I9026])
-::insert-2(I9030)
+::insert-2([http://partsregistry.org/Part:BBa_I9030 BBa_I9030])
 |}
 {|align="justify"
 |[[Image:Chiba-0901-2.JPG]]
 |
-::insert-3(S03154)
+::insert-3([http://partsregistry.org/Part:BBa_S03154 BBa_S03154])
 |}
 {|align="justify"
 |[[Image:Chiba-0901-3.JPG]]
 |
-::vector-4(R0010)
+::vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010])
 |}
@@ Line 141: / Line 141: @@
 :--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|zymo]]'''
-::insert-1(I9026) -> 7μL
+::insert-1([http://partsregistry.org/Part:BBa_I9026 BBa_I9026]) -> 7μL
-::insert-2(I9030) -> 7μL
+::insert-2([http://partsregistry.org/Part:BBa_I9030 BBa_I9030]) -> 7μL
-::insert-3(S03154) -> 7μL
+::insert-3([http://partsregistry.org/Part:BBa_S03154 BBa_S03154]) -> 7μL
-::vector-4(R0010) -> 15μL
+::vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010]) -> 15μL
 :--->'''[[Team:Chiba/protocol/ligation/dephosphorylation|SAP]]'''
-::vector-4(R0010)
+::vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010])
 :--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo]]'''
-::vector-4(R0010) -> 20μL
+::vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010]) -> 20μL
@@ Line 170: / Line 170: @@
 <tr>
 <td>TOTAL</td>
-<td>6</td>
+<td>6μl</td>
 </tr>
 </table>
 :From left;
-::*insert-1(I9026) -> OK
+::*insert-1([http://partsregistry.org/Part:BBa_I9026 BBa_I9026]) -> OK
-::*insert-2(I9030) -> OK
+::*insert-2([http://partsregistry.org/Part:BBa_I9030 BBa_I9030]) -> OK
-::*insert-3(S03154) -> None --> Transformation [http://partsregistry.org/Part:BBa_S03154 BBa_S03154]-->
+::*insert-3([http://partsregistry.org/Part:BBa_S03154 BBa_S03154]) -> None --> Transformation [http://partsregistry.org/Part:BBa_S03154 BBa_S03154]-->
 (2/9)'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''with [http://partsregistry.org/Part:BBa_K084009 BBa_K084009], [http://partsregistry.org/Part:BBa_K084010 BBa_K084010]
-::*vector-4(R0010)
+::*vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010])
 |}
@@ Line 189: / Line 189: @@
 </tr>
 <tr>
-<td>insert-1(I9026)</td>
+<td>insert-1([http://partsregistry.org/Part:BBa_I9026 BBa_I9026])</td>
 <td>3</td><td>-</td><td>3</td><td>-</td><td>-</td>
 </tr>
 <tr>
-<td>insert-2(I9030)</td>
+<td>insert-2([http://partsregistry.org/Part:BBa_I9030 BBa_I9030])</td>
 <td>-</td><td>3</td><td>-</td><td>3</td><td>-</td>
 </tr>
 <tr>
-<td>vector-4(R0010)</td>
+<td>vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010])</td>
 <td>3</td><td>3</td><td>-</td><td>-</td><td>3</td>
 </tr>
@@ Line 214: / Line 214: @@
 <tr>
 <td>TOTAL</td>
-<td>10</td><td>10</td><td>10</td><td>10</td><td>10</td>
+<td>10μl</td><td>10μl</td><td>10μl</td><td>10μl</td><td>10μl</td>
 </tr>
 </table>
@@ Line 241: / Line 241: @@
 :--->(2/9)'''Liquid Culture'''
 ===Team:Output===