Team:Chiba/Calendar-Home/21 August 2008

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*[http://partsregistry.org/Part:BBa_J22141 BBa_J22141](2007)
*[http://partsregistry.org/Part:BBa_J22141 BBa_J22141](2007)
-
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 20μl 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-
**(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°C freezer) in 2mL LB medium.(LB-Amp,Cm)(LB-Amp,Cm,AHL100nM)の2種類。
+
#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.
-
<br>37°C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。
+
#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.
-
<br>37°C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。
+
#We cultured for 12h at 37&deg;C.
-
<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。
+
#We iraddiated UV each plate. Wavelength:254nm,distance from the UV lamp to the cultures were 14cm.
-
<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。
+
#We performed the same operations on plates following 9 or 21h of UV
-
<br>37°C,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。
+
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.
 +
 
 +
#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.
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<table width="200" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="200" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<td width="257"></td>
<td width="257"></td>
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<td>AHLなし</td><td>AHL100nM</td>
+
<td>no AHL</td><td>AHL100nM</td>
<tr>
<tr>
<td>9h</td>
<td>9h</td>
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</tr>
</tr>
</table>
</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===
===Team:Communication===
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</tr>
</tr>
<tr>
<tr>
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<td>dNTP mix</td>
+
<td>dNTP mix(&mu;L)</td>
<td>10</td>
<td>10</td>
</tr>
</tr>
<tr>
<tr>
-
<td>Foward Primer</td>
+
<td>Foward Primer(&mu;L)</td>
<td>5</td>
<td>5</td>
</tr>
</tr>
<tr>
<tr>
-
<td>Reverse Primer</td>
+
<td>Reverse Primer(&mu;L)</td>
<td>5</td>
<td>5</td>
</tr>
</tr>
<tr>
<tr>
-
<td>DNA polymerase TAQ</td>
+
<td>DNA polymerase TAQ(&mu;L)</td>
<td>1</td>
<td>1</td>
</tr>
</tr>
<tr>
<tr>
-
<td>Thermopol Buffer</td>
+
<td>Thermopol Buffer(&mu;L)</td>
<td>5</td>
<td>5</td>
</tr>
</tr>
<tr>
<tr>
-
<td>dH<sub>2</sub>O</td>
+
<td>dH<sub>2</sub>O(&mu;L)</td>
<td>28</td>
<td>28</td>
</tr>
</tr>
<tr>
<tr>
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<td>TOTAL</td>
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<td>TOTAL(&mu;L)</td>
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<td>50μL</td>
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<td>50</td>
</tr>
</tr>
</table>
</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
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</tr>
</tr>
<tr>
<tr>
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<td>Loading Dye</td>
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<td>Loading Dye(&mu;L)</td>
<td>2</td>
<td>2</td>
</tr>
</tr>
<tr>
<tr>
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<td>dH<sub>2</sub>O</td>
+
<td>dH<sub>2</sub>O(&mu;L)</td>
<td>7</td>
<td>7</td>
</tr>
</tr>
<tr>
<tr>
-
<td>TOTAL</td>
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<td>TOTAL(&mu;L)</td>
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<td>12μL</td>
+
<td>12</td>
</tr>
</tr>
</table>
</table>

Latest revision as of 05:58, 30 October 2008

>Home | Notebook

20 August 2008 <|> 22 August 2008

Contents

Laboratory work

Team:Input

(-->20/8)

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

  • [http://partsregistry.org/Part:BBa_R0051 BBa_R0051](2007)
  • [http://partsregistry.org/Part:BBa_R0051 BBa_R0051](2006)
  • [http://partsregistry.org/Part:BBa_J06650 BBa_J06650](2007)
  • [http://partsregistry.org/Part:BBa_J06650 BBa_J06650](2006)
  • [http://partsregistry.org/Part:BBa_J22136 BBa_J22136](2007)
  • [http://partsregistry.org/Part:BBa_J22141 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-
  1. 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.
  2. We diluted 105-fold, and plated 20ul of the resulting solution to (LB-Amp,Cm)(LB-Amp,cm,AHL100nM) plates each other.
  3. We cultured for 12h at 37°C.
  4. We iraddiated UV each plate. Wavelength:254nm,distance from the UV lamp to the cultures were 14cm.
  5. We performed the same operations on plates following 9 or 21h of UV

exposure.

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

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

  1. 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 AHLAHL100nM
9h 606453
12h 146151

result
GFP fluorescence was observed from plates without AHL.

Team:Communication

(20/8)--->PCR
  • [http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2007)
  • [http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2006)
  • [http://partsregistry.org/Part:BBa_C0076 BBa_C0076](2007)
  • [http://partsregistry.org/Part:BBa_C0078 BBa_C0078](2007)
  • [http://partsregistry.org/Part:BBa_C0078 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
Chiba-0821.JPG
Sample DNA 3
Loading Dye(μL) 2
dH2O(μL) 7
TOTAL(μL) 12
From left;
  • [http://partsregistry.org/Part:BBa_C0078 BBa_C0078](2007) -> None
  • [http://partsregistry.org/Part:BBa_C0078 BBa_C0078](2006) -> None
  • [http://partsregistry.org/Part:BBa_C0076 BBa_C0076](2007) -> None
  • [http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2007) -> OK
  • [http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2006) -> OK


Transformation
competent cells : XL10G
  • [http://partsregistry.org/Part:BBa_S03154 BBa_S03154](2007)
  • [http://partsregistry.org/Part:BBa_S03154 BBa_S03154](2006)
  • [http://partsregistry.org/Part:BBa_I9026 BBa_I9026](2007)
  • [http://partsregistry.org/Part:BBa_I9026 BBa_I9026](2006)
  • [http://partsregistry.org/Part:BBa_I9030 BBa_I9030](2007)
  • [http://partsregistry.org/Part:BBa_I9030 BBa_I9030](2006)



--->(23/8)Digestion

Team:Output

Transformation

  • [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](2007)-->colony
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