Team:Chiba/Calendar-Home/21 August 2008

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Latest revision as of 05:58, 30 October 2008

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20 August 2008 <|> 22 August 2008

Laboratory work

Team:Input

(-->20/8)

Inoculated transformants for 12 hours in LB 2mL containing Ampicillin.

BBa_R0051(2007)
BBa_R0051(2006)
BBa_J06650(2007)
BBa_J06650(2006)
BBa_J22136(2007)
BBa_J22141(2007)

BBa_J22141:After inoculated for 11 hours, added 20μl IPTG and cultured one hour.

UV irradiation test (plate phase)
two plasmids from(JW1908)
- (Repressor)-Ptrc-LuxR-Plux-cI-colE1-Amp-
- (Reporter)-PcI-GFP-p15a-Cm-

Inoculated (Reporter) culture from glycerol stock(-80°C freezer) in 2mL LB-Ampicillin, Chloramphenicol medium and LB-Ampicillin, Chloramphenicol, 100nmAHL mediumfor 12h, at 37°C.
We diluted 10⁵-fold, and plated 20ul of the resulting solution to (LB-Amp,Cm)(LB-Amp,cm,AHL100nM) plates each other.
We cultured for 12h at 37°C.
We iraddiated UV each plate. Wavelength:254nm,distance from the UV lamp to the cultures were 14cm.
We performed the same operations on plates following 9 or 21h of UV

exposure.

Colonies from both plates were picked and cultured in 2mL of

LB-Amp,Cm or LB-Amp, Cm, 100nMAHL for 12h at 37°C.

We diluted 10⁵-fold, and plated 20ul of the

resulting solution to (LB-Amp,Cm)(LB-Amp,cm,AHL100nM) plates each other.

Colony count

	no AHL	AHL100nM
9h	606	453
12h	146	151

result
GFP fluorescence was observed from plates without AHL.

Team:Communication

(20/8)--->PCR

BBa_C0070(2007)
BBa_C0070(2006)
BBa_C0076(2007)
BBa_C0078(2007)
BBa_C0078(2006)

DNA Template	1
dNTP mix(μL)	10
Foward Primer(μL)	5
Reverse Primer(μL)	5
DNA polymerase TAQ(μL)	1
Thermopol Buffer(μL)	5
dH₂O(μL)	28
TOTAL(μL)	50

95°C,5min -> ( 95°C,1min -> 52°C,1min -> 72°C,1min )･･･25cycles -> 72°C,10min -> 6°C

--->Gel Check

Sample DNA	3
Loading Dye(μL)	2
dH₂O(μL)	7
TOTAL(μL)	12

From left;

BBa_C0078(2007) -> None
BBa_C0078(2006) -> None
BBa_C0076(2007) -> None
BBa_C0070(2007) -> OK
BBa_C0070(2006) -> OK

Transformation

competent cells : XL10G

BBa_S03154(2007)
BBa_S03154(2006)
BBa_I9026(2007)
BBa_I9026(2006)
BBa_I9030(2007)
BBa_I9030(2006)

--->(23/8)Digestion

Team:Output

Transformation

BBa_J32007(2007)-->no colony-->no colony(2 times)-->colony(3times)
BBa_B0034(2007)-->colony

@@ Line 5: / Line 5: @@
 ==Laboratory work==
 ===Team:Input===
-(-->20/8)pick transformants
+(-->20/8)
+Inoculated transformants for 12 hours in LB 2mL containing Ampicillin.
 *[http://partsregistry.org/Part:BBa_R0051 BBa_R0051](2007)
@@ Line 14: / Line 16: @@
 *[http://partsregistry.org/Part:BBa_J22141 BBa_J22141](2007)
-を２ml培養、12h。ただしコロニーが１個だけだったBBa_J22141は2ml培養の11h後にIPTGを20μl加え、
+BBa_J22141:After inoculated for 11 hours, added 20&mu;l IPTG and cultured one hour.
-さらに1h培養した。
-*UV照射実験(プレート編)(~24日)
+*UV irradiation test  (plate phase)
-*two plasmids from ---
+*two plasmids from(JW1908)
 **(Repressor)-Ptrc-LuxR-Plux-cI-colE1-Amp-
-**(Reporter)-PcI-GFP-p15a-Cm-(BW)
+**(Reporter)-PcI-GFP-p15a-Cm-
+#Inoculated (Reporter) culture from glycerol stock(-80&deg;C freezer) in 2mL LB-Ampicillin, Chloramphenicol medium and LB-Ampicillin, Chloramphenicol, 100nmAHL mediumfor 12h, at 37&deg;C.
+#We diluted 10<sup>5</sup>-fold, and plated 20ul of the resulting solution to (LB-Amp,Cm)(LB-Amp,cm,AHL100nM) plates each other.
+#We cultured for 12h at 37&deg;C.
+#We iraddiated UV each plate. Wavelength:254nm,distance from the UV lamp to the cultures were 14cm.
+#We performed the same operations on plates following 9 or 21h of UV
+exposure.
+#Colonies from both plates were picked and cultured in 2mL of
+LB-Amp,Cm or LB-Amp, Cm, 100nMAHL for 12h at 37&deg;C.
-#Inoculated (Reporter) culture from glycerol stock(-80℃ freezer) in 2mL LB medium.(LB-Amp,Cm)(LB-Amp,Cm,AHL100nM)の2種類。
+#We diluted 10<sup>5</sup>-fold, and plated 20ul of the
-<br>37℃で12h培養後、濁ったらそれぞれを10<sup>5</sup>希釈してAHLが入っていないものはLB-Amp,Cm,AHLが入っているものはAHL-Amp,Cm,AHL100nMのプレートにそれぞれ20μl撒く。
+resulting solution to (LB-Amp,Cm)(LB-Amp,cm,AHL100nM) plates each other.
-<br>37℃で12h培養後、きちんとコロニーができていることを確認し、両方のプレートにUVを照射する。UVの波長は254nm,UVランプとプレートの距離は14cm。
-<br>UVを照射して9h後、21h後にそれぞれ両方のプレートに次の同じ操作をする。
-<br>両方のプレートのコロニーをつついてそれぞれ(LB-Amp,Cm),(LB-Amp,Cm,AHL100nM)2ml培養する。
-<br>37℃,で12h培養後、濁っていたので、それぞれを(LB-Amp,Cm),(LB-Amp,Cm,AHL100nM)のプレートに10<sup>5</sup>希釈して20μl撒いた。
@@ Line 34: / Line 40: @@
 <table width="200" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
 <td width="257"></td>
-<td>AHLなし</td><td>AHL100nM</td>
+<td>no AHL</td><td>AHL100nM</td>
 <tr>
 <td>9h</td>
@@ Line 44: / Line 50: @@
 </tr>
 </table>
+result
+<BR>
-AHLが入っていないほうのコロニーはいづれも光っていた。→機能チェックはOK
+GFP fluorescence was observed from plates without AHL.
 ===Team:Communication===
@@ Line 63: / Line 69: @@
 </tr>
 <tr>
-<td>dNTP mix</td>
+<td>dNTP mix(&mu;L)</td>
 <td>10</td>
 </tr>
 <tr>
-<td>Foward Primer</td>
+<td>Foward Primer(&mu;L)</td>
 <td>5</td>
 </tr>
 <tr>
-<td>Reverse Primer</td>
+<td>Reverse Primer(&mu;L)</td>
 <td>5</td>
 </tr>
 <tr>
-<td>DNA polymerase TAQ</td>
+<td>DNA polymerase TAQ(&mu;L)</td>
 <td>1</td>
 </tr>
 <tr>
-<td>Thermopol Buffer</td>
+<td>Thermopol Buffer(&mu;L)</td>
 <td>5</td>
 </tr>
 <tr>
-<td>dH<sub>2</sub>O</td>
+<td>dH<sub>2</sub>O(&mu;L)</td>
 <td>28</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(&mu;L)</td>
-<td>50μL</td>
+<td>50</td>
 </tr>
 </table>
-:95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃
+:95°C,5min -> ( 95°C,1min -> 52°C,1min -> 72°C,1min )･･･25cycles -> 72°C,10min -> 6°C
@@ Line 107: / Line 113: @@
 </tr>
 <tr>
-<td>Loading Dye</td>
+<td>Loading Dye(&mu;L)</td>
 <td>2</td>
 </tr>
 <tr>
-<td>dH<sub>2</sub>O</td>
+<td>dH<sub>2</sub>O(&mu;L)</td>
 <td>7</td>
 </tr>
 <tr>
-<td>TOTAL</td>
+<td>TOTAL(&mu;L)</td>
-<td>12μL</td>
+<td>12</td>
 </tr>
 </table>
@@ Line 146: / Line 152: @@
 ===Team:Output===
 [[Team:Chiba/protocol/transformation|Transformation]]
-*[http://partsregistry.org/Part:BBa_J32007 BBa_J32007](Lux CDABE)-->no colony
+*[http://partsregistry.org/Part:BBa_J32007 BBa_J32007](2007)-->no colony-->no colony(2 times)-->colony(3times)
-*[http://partsregistry.org/Part:BBa_B0034 BBa_B0034](RBS)-->colony
+*[http://partsregistry.org/Part:BBa_B0034 BBa_B0034](2007)-->colony
-:結果:成功→RBS
-:失敗→Lux CDABE(二回行ったがどちらもコロニーができなかった)→三回目に成功