Team:Chiba/Calendar-Home/21 August 2008

From 2008.igem.org

(Difference between revisions)
(→Team:Input)
(→Team:Communication)
 
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</tr>
</tr>
<tr>
<tr>
-
<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>
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<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>
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</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>
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</tr>
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<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>
+
<td>TOTAL(&mu;L)</td>
-
<td>12μL</td>
+
<td>12</td>
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</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.

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


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;


Transformation
competent cells : XL10G



--->(23/8)Digestion

Team:Output

Transformation

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