Team:Chiba/Notebook/Calendar/24 September 2009

From 2009.igem.org

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-
== Examine limit of AHL generation ==
+
== Examine limit of AHL generation(1)-3 ==
Yesterday's operation is [https://2009.igem.org/wiki/index.php?title=Team:Chiba/Notebook/Calendar/23_September_2009 here].  
Yesterday's operation is [https://2009.igem.org/wiki/index.php?title=Team:Chiba/Notebook/Calendar/23_September_2009 here].  
-
22:00
+
*Today's operation
 +
14:00
-
We measured OD.
+
We made plates from each of mixture and 10 mL of LB-Amp, Cm solution medium.
-
OD<sub>600</sub>=1.98
 
 +
*Element of mixtures
-
Then, we spun down this culture solution and add 12.5 mL of flesh LB-Amp.
+
<table width="900" border="1" cellpadding="0"  cellspacing="0" bordercolor="#000000"><tr>
 +
<td width="100">Sample Namber</td>
 +
<td width="100">1</td>
 +
<td width="100">2</td>
 +
<td width="100">3</td>
 +
<td width="100">4</td>
 +
<td width="100">5</td>
 +
<td width="100">6</td>
 +
<td width="100">7</td>
 +
<td width="100">8</td>
 +
</tr>
 +
<tr>
 +
<td>supernatant solution</td>
 +
<td>5 mL</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>20 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>1</td>
 +
<td>---</td>
 +
<td>5 mL</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
</tr>
 +
<tr>
 +
<td>2</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>5 mL</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>5 mL</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
</tr>
 +
<tr>
 +
<td>4</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>5 mL</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
</tr>
 +
<tr>
 +
<td>5</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>5 mL</td>
 +
<td>---</td>
 +
<td>---</td>
 +
</tr>
 +
<tr>
 +
<td>6</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>---</td>
 +
<td>5 mL</td>
 +
<td>---</td>
 +
</tr>
 +
<tr>
 +
<td>LB-Amp,Cm(liquid)</td>
 +
<td>10 mL</td>
 +
<td>10 mL</td>
 +
<td>10 mL</td>
 +
<td>10 mL</td>
 +
<td>10 mL</td>
 +
<td>10 mL</td>
 +
<td>10 mL</td>
 +
<td>9.98 mL</td>
 +
</tr>
 +
<tr>
 +
<td>Total</td>
 +
<td>10 mL</td>
 +
<td>10 mL</td>
 +
<td>10 mL</td>
 +
<td>10 mL</td>
 +
<td>10 mL</td>
 +
<td>10 mL</td>
 +
<td>10 mL</td>
 +
<td>10 mL</td>
 +
</tr>
 +
</table>
 +
== Transformation(2)-2 ==
 +
Yesterday's operation is [https://2009.igem.org/Team:Chiba/Notebook/Calendar/23_September_2009 here].
-
22:10
 
-
We add 12.5 &mu;L of IPTG to the culture solution and started culture at 37 degrees Celsius.
+
 
 +
*Today's operation
 +
11:15-
 +
 
 +
We picked colony(plux-GFP only) and cultured it.
 +
 
 +
 
 +
 
 +
15:50-
 +
 
 +
Main culture
 +
 
 +
 
 +
20:30-
 +
 
 +
Mini prep.
 +
 
 +
And resultant DNA was saved in freezer.
 +
 
 +
 
 +
 
 +
== Transformation(3)-1 ==
 +
*Plasmids
 +
ptet-GFP pSB1A2 [http://partsregistry.org/Part:BBa_I13522 BBa_I13522](Amp)
 +
 
 +
ptet-RFP pSB1A3 [http://partsregistry.org/Part:BBa_I13521 BBa_I13521](Amp)
 +
 
 +
ptet-CFP pSB1A2 [http://partsregistry.org/Part:BBa_I13600 BBa_I13600](Amp)
 +
 
 +
mRFP without Nco1 site(Cm)
 +
 
 +
 
 +
*Competent Cells
 +
[https://2009.igem.org/wiki/index.php?title=Team:Chiba/Notebook/Calendar/23_September_2009 JW1262]
 +
 
 +
 
 +
21:50-
 +
 
 +
We cultured each plates.
 +
 
 +
 
 +
 
 +
== Digestion Test ==
 +
*Samples
 +
1 : ECFP([http://partsregistry.org/Part:BBa_I6057 BBa_I6057])
 +
 
 +
2 : mCherry([http://partsregistry.org/Part:BBa_E2060 BBa_E2060])
 +
 
 +
3 : V-YFP([http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
 +
 
 +
4 : mOrange([http://partsregistry.org/Part:BBa_E2050 BBa_E2050])
 +
 
 +
5 : LacZ &alpha;([http://partsregistry.org/Part:BBa_T9003 BBa_T9003])
 +
 
 +
6 : mRFP without Nco1 site
 +
 
 +
*Doble Digestion
 +
<table width="100" border="1" cellpadding="0"  cellspacing="0" bordercolor="#000000"><tr>
 +
<td width="50">Sample</td>
 +
<td width="50">3 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>EcoR1</td>
 +
<td>0.20 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Pst1</td>
 +
<td>0.20 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Buffer</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>BSA</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>dw</td>
 +
<td>4.6 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Total</td>
 +
<td>10 &mu;L</td>
 +
</tr>
 +
</table>
 +
 
 +
 
 +
 
 +
*Doble Digestion's Master Mix
 +
<table width="100" border="1" cellpadding="0"  cellspacing="0" bordercolor="#000000"><tr>
 +
<td width="50">Sample</td>
 +
<td width="50">3 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>EcoR1</td>
 +
<td>1.4 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Pst1</td>
 +
<td>1.4 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Buffer</td>
 +
<td>7 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>BSA</td>
 +
<td>7 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>dw</td>
 +
<td>32.2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Total</td>
 +
<td>49 &mu;L</td>
 +
</tr>
 +
</table>
 +
 
 +
 
 +
*Single Digestion
 +
<table width="100" border="1" cellpadding="0"  cellspacing="0" bordercolor="#000000"><tr>
 +
<td width="50">Sample</td>
 +
<td width="50">3 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>EcoR1</td>
 +
<td>0.20 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Buffer</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>dw</td>
 +
<td>6.8 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Total</td>
 +
<td>10 &mu;L</td>
 +
</tr>
 +
</table>
 +
 
 +
 
 +
 
 +
*Single Digestion's Master Mix
 +
<table width="100" border="1" cellpadding="0"  cellspacing="0" bordercolor="#000000"><tr>
 +
<td width="50">EcoR1</td>
 +
<td width="50">1.4 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Buffer</td>
 +
<td>7 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>dw</td>
 +
<td>40.6 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Total</td>
 +
<td>49 &mu;L</td>
 +
</tr>
 +
</table>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
12:50
 +
 
 +
Warm up at 37 degrees Celsius
 +
 
 +
 
 +
13:30
 +
 
 +
Gel electrophoretic analysis
 +
 
 +
== To judge character of LuxR mutants(3)-1 ==
 +
We poured 1 mL of LB-Amp, Cm liquid medium in 96 deep well and added glycerol stocks.
 +
 
 +
 
 +
22:10-
 +
 
 +
We cultured and shook it at 37 degrees Celsius.

Latest revision as of 08:03, 3 October 2009

>Go to the Notebook page

(23_September_2009 <|>25_September_2009)


Contents

Examine limit of AHL generation(1)-3

Yesterday's operation is here.


  • Today's operation

14:00

We made plates from each of mixture and 10 mL of LB-Amp, Cm solution medium.


  • Element of mixtures
Sample Namber 1 2 3 4 5 6 7 8
supernatant solution 5 mL --- --- --- --- --- --- 20 μL
1 --- 5 mL --- --- --- --- --- ---
2 --- --- 5 mL --- --- --- --- ---
3 --- --- --- 5 mL --- --- --- ---
4 --- --- --- --- 5 mL --- --- ---
5 --- --- --- --- --- 5 mL --- ---
6 --- --- --- --- --- --- 5 mL ---
LB-Amp,Cm(liquid) 10 mL 10 mL 10 mL 10 mL 10 mL 10 mL 10 mL 9.98 mL
Total 10 mL 10 mL 10 mL 10 mL 10 mL 10 mL 10 mL 10 mL

Transformation(2)-2

Yesterday's operation is here.


  • Today's operation

11:15-

We picked colony(plux-GFP only) and cultured it.


15:50-

Main culture


20:30-

Mini prep.

And resultant DNA was saved in freezer.


Transformation(3)-1

  • Plasmids

ptet-GFP pSB1A2 [http://partsregistry.org/Part:BBa_I13522 BBa_I13522](Amp)

ptet-RFP pSB1A3 [http://partsregistry.org/Part:BBa_I13521 BBa_I13521](Amp)

ptet-CFP pSB1A2 [http://partsregistry.org/Part:BBa_I13600 BBa_I13600](Amp)

mRFP without Nco1 site(Cm)


  • Competent Cells

JW1262


21:50-

We cultured each plates.


Digestion Test

  • Samples

1 : ECFP([http://partsregistry.org/Part:BBa_I6057 BBa_I6057])

2 : mCherry([http://partsregistry.org/Part:BBa_E2060 BBa_E2060])

3 : V-YFP([http://partsregistry.org/Part:BBa_K084003 BBa_K084003])

4 : mOrange([http://partsregistry.org/Part:BBa_E2050 BBa_E2050])

5 : LacZ α([http://partsregistry.org/Part:BBa_T9003 BBa_T9003])

6 : mRFP without Nco1 site

  • Doble Digestion
Sample 3 μL
EcoR1 0.20 μL
Pst1 0.20 μL
Buffer 1 μL
BSA 1 μL
dw 4.6 μL
Total 10 μL


  • Doble Digestion's Master Mix
Sample 3 μL
EcoR1 1.4 μL
Pst1 1.4 μL
Buffer 7 μL
BSA 7 μL
dw 32.2 μL
Total 49 μL


  • Single Digestion
Sample 3 μL
EcoR1 0.20 μL
Buffer 1 μL
dw 6.8 μL
Total 10 μL


  • Single Digestion's Master Mix
EcoR1 1.4 μL
Buffer 7 μL
dw 40.6 μL
Total 49 μL



12:50

Warm up at 37 degrees Celsius


13:30

Gel electrophoretic analysis

To judge character of LuxR mutants(3)-1

We poured 1 mL of LB-Amp, Cm liquid medium in 96 deep well and added glycerol stocks.


22:10-

We cultured and shook it at 37 degrees Celsius.

Retrieved from "http://2009.igem.org/Team:Chiba/Notebook/Calendar/24_September_2009"