Team:Chiba/jk/β

From 2008.igem.org

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-
>[[Team:Chiba/jk|実験ログ]]<Br>
+
<html><link rel="stylesheet" href="https://2008.igem.org/wiki/index.php?title=User:Maiko/chiba.css&action=raw&ctype=text/css" type="text/css" /></html>
 +
>[[Team:Chiba/Internal|ノートに戻る]]
-
'''β班の実験ノート'''<Br>
 
-
[[Team:Chiba/jk/β/weaks 1-2|weeks 1-2]]<Br>
 
-
[[Team:Chiba/jk/β/weaks 3-4|weeks 3-4]]<Br>
 
-
==Week 1==
+
==送受信班の実験ノート==
 +
*[[Team:Chiba/jk/β/week 1|week 1]]
 +
*[[Team:Chiba/jk/β/week 2|week 2]]
 +
*[[Team:Chiba/jk/β/week 3|week 3]]
 +
*[[Team:Chiba/jk/β/week 4|week 4]]
 +
*[[Team:Chiba/jk/β/week 5|week 5]]
 +
*[[Team:Chiba/jk/β/week 6|week 6]]
-
===17 August, 2008===  
+
==モジュール解説==
-
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
+
===感度差による応答時間の差===
-
:competent cells : XL10G
+
任意の順番と、delay-timeをもった遺伝子発現を実現するために、バクテリア間の細胞間通信、クオラムセンシングを利用する。クオラムセンシングでは、応答閾値を超えるAHLが蓄積されたとき初めて、目的遺伝子の発現がおこる。AHL情報がじゅうぶんゆっくり蓄積するとき、ひとつのAHL送信装置に対して、感度の異なる複数の受信装置によってさまざまな出力装置を独立に起動すれば、感度の高いAHL 受信機ほど先に起動する。
-
::*BBa_R0040(2008) [http://partsregistry.org/Part:BBa_R0040] -> No colonies on plates
+
-
::*BBa_C0070(2008) [http://partsregistry.org/Part:BBa_C0070] -> No colonies on plates
+
-
::*BBa_C0076(2008) [http://partsregistry.org/Part:BBa_C0076] -> No colonies on plates
+
-
::*BBa_C0077(2008) [http://partsregistry.org/Part:BBa_C0077] -> No colonies on plates
+
-
::*BBa_C0078(2008) [http://partsregistry.org/Part:BBa_C0078] -> No colonies on plates
+
-
::*BBa_C0079(2008) [http://partsregistry.org/Part:BBa_C0079] -> No colonies on plates
+
-
===18 August, 2008===  
+
=== Controlling the Delay time of the AHL signaling ===
-
:'''[[Team:Chiba/protocol/PCR|PCR]]'''
+
LuxIのつくるAHLに対して、感度差による応答時間差は、２つの方法で実現することができる。
-
::*BBa_C0070(2008) [http://partsregistry.org/Part:BBa_C0070]
+
#LuxR/ Pluxの感度変異体を用いる場合<br>LuxRの変異体を用いれば、pluxがonになるAHL濃度が<1nMから~10nMまで変化する。[http://authors.library.caltech.edu/5553/ C. H. Collins.''et al.:''Mol.Microbiol.2005.'''55'''(3).712–723]
-
::*BBa_C0076(2008) [http://partsregistry.org/Part:BBa_C0076]
+
#クロストークを使う場合
-
::*BBa_C0077(2008) [http://partsregistry.org/Part:BBa_C0077]
+
:AHL=Acyl-homoserine Lactoneには、そのアシル基が異なる種類がある。luxIが合成するAHLは、3-oxo-hyxanoyl-Homoserine Lactone (=OHHL)であり、3-oxo-dodecanoyl-homoserine Lactone(=ODHL)や、butyryl-homoserine Lactone(=HHL)が存在する。それぞれ、合成酵素はLasI、RhlI（ともにシュードモナス由来）と呼ばれる。このほかのAHL分子と、その合成酵素を以下に示した。これらのAHL分子は、アシル基のみが異なるため、一定の割合でLuxRと相互作用する（cross-talkする）。たとえば、OdHLとLuxRとのcross-talk（LasI-LuxR）は、OHHLとLuxRの場合（LuxI-LuxR）の1/20である。AHL分子の蓄積速度が一定ならば，luxI-LuxR/Pluxよりも20倍遅くスイッチが入ることとなる。
-
::*BBa_C0078(2008) [http://partsregistry.org/Part:BBa_C0078]
+
-
::*BBa_C0079(2008) [http://partsregistry.org/Part:BBa_C0079]
+
-
::*BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]･･･control
+
-
 
+
*AHLsおよびその合成酵素
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+
<table width="500" border="1" cellpadding="0" cellspacing="0" bordercolor="#000000" center>
<tr>
<tr>
-
<td width="257">DNA Template</td>
+
<td width="200">Strain</td>
-
<td>1</td>
+
<td width="200">Signal Molecule</td>
 +
<td width="150">Enzyme<td>
</tr>
</tr>
<tr>
<tr>
-
<td>dNTP mix</td>
+
<td>P.aeruginosa</td>
-
<td>10</td>
+
<td>C4-HSL</td>
 +
<td>RhlI</td>
</tr>
</tr>
<tr>
<tr>
-
<td>Foward Primer</td>
+
<td>V. fisheri</td>
-
<td>5</td>
+
<td>C6-3-oxo-HSL</td>
 +
<td>LuxI</td>
</tr>
</tr>
<tr>
<tr>
-
<td>Reverse Primer</td>
+
<td>A.tumefaciens</td>
-
<td>5</td>
+
<td>C8-3-oxo-HSL</td>
 +
<td>TraI</td>
</tr>
</tr>
<tr>
<tr>
-
<td>DNA polymerase</td>
+
<td>P.aeruginosa</td>
-
<td>1</td>
+
<td>C10-3-oxo-HSL</td>
 +
<td>VanI</td>
</tr>
</tr>
-
<tr>
 
-
<td>Thermopol Buffer</td>
 
-
<td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>68</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>100μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃
 
-
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0818.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
<tr>
<tr>
-
<td width="257">Sample DNA</td>
+
<td>P.aeruginosa</td>
-
<td>3</td>
+
<td>C12-3-oxo-HSL</td>
 +
<td>LasI</td>
</tr>
</tr>
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:From left;
 
-
::*BBa_C0070(2008) [http://partsregistry.org/Part:BBa_C0070] ->None
 
-
::*BBa_C0076(2008) [http://partsregistry.org/Part:BBa_C0076] ->None
 
-
::*BBa_C0077(2008) [http://partsregistry.org/Part:BBa_C0077] ->None
 
-
::*BBa_C0078(2008) [http://partsregistry.org/Part:BBa_C0078] ->None
 
-
::*BBa_C0079(2008) [http://partsregistry.org/Part:BBa_C0079] ->None
 
-
::*BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]･･･control ->OK
 
-
|}
 
-
 
-
 
-
 
-
 
-
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
competent cells : XL10G
 
-
 
-
::*BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070]
 
-
::*BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070]
 
-
::*BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076]
 
-
::*BBa_C0076(2006) [http://partsregistry.org/Part:BBa_C0076] -> No colonies on plates(19/8)
 
-
::*BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078]
 
-
::*BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078]
 
-
::*BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170]
 
-
::*BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170]
 
-
::*BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178]
 
-
::*BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178]
 
-
 
-
 
-
:--->(20/8)[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]
 
-
 
-
===19 August, 2008===
 
-
:'''[[Team:Chiba/protocol/PCR|PCR]]'''
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_S3154(2007) [http://partsregistry.org/Part:BBa_S3154]
 
-
::*BBa_S3154(2006) [http://partsregistry.org/Part:BBa_S3154]
 
-
::*BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]･･･control
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
<tr>
<tr>
-
<td width="257">DNA Template</td>
+
<td>R.leguminosarum</td>
-
<td>1</td>
+
<td>C14-3-hydroxy-HSL</td>
-
</tr>
+
<td>CinI</td>
-
<tr>
+
-
<td>dNTP mix</td>
+
-
<td>10</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Foward Primer</td>
+
-
<td>5</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Reverse Primer</td>
+
-
<td>5</td>
+
-
</tr>
+
-
<tr>
+
-
<td>DNA polymerase VENT</td>
+
-
<td>1</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Thermopol Buffer</td>
+
-
<td>5</td>
+
-
</tr>
+
-
<tr>
+
-
<td>dH2O</td>
+
-
<td>28</td>
+
-
</tr>
+
-
<tr>
+
-
<td>TOTAL</td>
+
-
<td>50μL</td>
+
</tr>
</tr>
</table>
</table>
 +
[http://partsregistry.org/Part:BBa_F2620:Specificity AHLsについて]
 +
==実験==
 +
===Construction(From BioBrick)===
 +
:*Sender(Autoinducer synthase)
 +
::LuxI,LasI,CinI,RhlI(lacプロモーター)
 +
:*Receiver
 +
::LuxR,LasR,CinR,RhlR
-
:95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃
 
-
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0819.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:From left;
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026] ->None
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026] ->None
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030] ->None
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030] ->None
 
-
::*BBa_S3154(2007) [http://partsregistry.org/Part:BBa_S3154] ->None
 
-
::*BBa_S3154(2006) [http://partsregistry.org/Part:BBa_S3154] ->None
 
-
::*BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]･･･control  ->OK
 
-
|}
 
-
 
-
 
-
 
-
===20 August, 2008===
 
-
:(18/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
::*BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070]
 
-
::*BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070]
 
-
::*BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076]
 
-
::*BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078]
 
-
::*BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078]
 
-
::*BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170]
 
-
::*BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170]
 
-
::*BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178]
 
-
::*BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178]
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0820-3.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:From left;
 
-
::*BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076] -> None
 
-
::*BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078] -> None
 
-
::*BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078] -> None
 
-
|}
 
-
 
-
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0820-4.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>24μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:From left;
 
-
::*BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078] -> None
 
-
::*BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078] -> OK
 
-
::*BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076] -> None
 
-
::*BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070] -> OK
 
-
::*BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070] -> OK
 
-
|}
 
-
 
-
 
-
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0820.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:From left;<br>
 
-
::*BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -> OK<br>
 
-
::*BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] -> OK<br>
 
-
::*BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] -> OK<br>
 
-
::*BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] -> OK<br>
 
-
|}
 
-
 
-
 
-
--->'''[[Team:Chiba/protocol/digestion|Digestion test]]'''
 
-
::*BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170]
 
-
::*BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170]
 
-
::*BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178]
 
-
::*BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178]
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td>6</td><td>7</td><td>8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170]</td>
 
-
<td>3</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170]</td>
 
-
<td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178]</td>
 
-
<td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178]</td>
 
-
<td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>EcoRⅠ</td>
 
-
<td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>PstⅠ</td>
 
-
<td>-</td><td>-</td><td>-</td><td>-</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer EcoRⅠ</td>
 
-
<td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>4.9</td><td>4.9</td><td>4.9</td><td>4.9</td><td>4.8</td><td>4.8</td><td>4.8</td><td>4.8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0820-2.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12μL</td>
 
-
</tr>
 
-
</table>
 
-
:From left;
 
-
:(Single Digestion 1~4)
 
-
::*BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] -> OK
 
-
::*BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -> OK
 
-
::*BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] -> OK
 
-
::*BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] -> OK
 
-
:(Double Digestion 1~4)
 
-
::*BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] -> OK
 
-
::*BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -> OK
 
-
::*BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] -> OK
 
-
::*BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] -> OK
 
-
|}
 
-
 
-
===21 August, 2008===
 
-
:(20/8)--->'''[[Team:Chiba/protocol/PCR|PCR]]'''
 
-
::*BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070]
 
-
::*BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070]
 
-
::*BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076]
 
-
::*BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078]
 
-
::*BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078]
 
-
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">DNA Template</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dNTP mix</td>
 
-
<td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Foward Primer</td>
 
-
<td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Reverse Primer</td>
 
-
<td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>DNA polymerase TAQ</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Thermopol Buffer</td>
 
-
<td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>28</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>50μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
:95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃
 
-
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0821.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:From left;
 
-
::*BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078] -> None
 
-
::*BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078] -> None
 
-
::*BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076] -> None
 
-
::*BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070] -> OK
 
-
::*BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070] -> OK
 
-
|}
 
-
 
-
 
-
 
-
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
:competent cells : XL10G
 
-
::*BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
 
-
 
-
 
-
 
-
 
-
--->(23/8)'''[[Team:Chiba/protocol/digestion|Digestion]]'''
 
-
 
-
 
-
 
-
===22 August, 2008===
 
-
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
:competent cells : XL10G
 
-
::*BBa_J13002(2007) [http://partsregistry.org/Part:BBa_J13002]
 
-
::*BBa_J13002(2006) [http://partsregistry.org/Part:BBa_J13002]
 
-
::*BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]
 
-
::*BBa_J04500(2006) [http://partsregistry.org/Part:BBa_J04500]
 
-
:(Locate & 2 Punch ~iyama ver.~)
 
-
::*BBa_C0076(2008) [http://partsregistry.org/Part:BBa_C0076] -> No colonies on plates
 
-
::*BBa_C0077(2008) [http://partsregistry.org/Part:BBa_C0077] -> No colonies on plates
 
-
::*BBa_C0078(2008) [http://partsregistry.org/Part:BBa_C0078] -> No colonies on plates
 
-
::*BBa_J04500(2008) [http://partsregistry.org/Part:BBa_J04500] -> No colonies on plates
 
-
 
-
 
-
 
-
--->(24/8)'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
 
-
===23 August, 2008===
 
-
:(21/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
::*BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/digestion|Digestion test]]'''
 
-
::*BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]
 
-
 
-
 
-
:<table width="500" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="270">Sample No</td>
 
-
<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td>10</td><td>11</td><td>12</td><td>13</td><td>14</td><td>15</td><td>16</td><td>17</td><td>18</td><td>19</td><td>20</td><td>21</td>
 
-
</tr><tr>
 
-
<td>BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]</td>
 
-
<td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]</td>
 
-
<td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]</td>
 
-
<td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]</td>
 
-
<td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<tr>
 
-
<td>BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]</td>
 
-
<td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td></tr>
 
-
<tr>
 
-
<td>BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]</td>
 
-
<td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td></tr>
 
-
<tr>
 
-
<td>BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]</td>
 
-
<td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td></tr>
 
-
<tr>
 
-
<td>EcoRⅠ</td>
 
-
<td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>PstⅠ</td>
 
-
<td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer EcoRⅠ</td>
 
-
<td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0.9</td><td>0.9</td><td>0.9</td><td>0.9</td><td>0.9</td><td>0.9</td><td>0.9</td><td>0.8</td><td>0.8</td><td>0.8</td><td>0.8</td><td>0.8</td><td>0.8</td><td>0.8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td><td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>1</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
 
-
:--->[[Team:Chiba/protocol/gelcheck|Gel Check]]
 
-
{|align="justify"
 
-
|[[Image:Chiba-0823-1.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1~6</td><td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>3</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>2</td><td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td><td>6</td>
 
-
</tr>
 
-
</table>
 
-
:From left; Sanple No. 1~7
 
-
::*BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]
 
-
|}
 
-
 
-
 
-
 
-
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0823-2.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>8~21</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:From left;
 
-
:(Single Digestion) Sanple No. 8~14
 
-
::*BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154] -> OK
 
-
::*BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154] -> OK
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026] -> OK
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026] -> OK
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030] -> OK
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030] -> OK
 
-
::*BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500] -> OK
 
-
 
-
:(Double Digestion) Sample No. 15~21
 
-
::*BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154] -> OK
 
-
::*BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154] -> OK
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026] -> OK
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026] -> OK
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030] -> OK
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030] -> OK
 
-
::*BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500] -> OK?
 
-
|}
 
-
 
-
 
-
 
-
 
-
:'''[[Team:Chiba/protocol/digestion|Digestion test]]'''
 
-
::BBa_J04500(2007:old-sample)[http://partsregistry.org/Part:BBa_J04500]
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1</td><td>2</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>3</td><td>3</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>EcoRⅠ</td>
 
-
<td>-</td><td>0.1</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SpeⅠ</td>
 
-
<td>-</td><td>-</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>EcoRⅠ Buffer</td>
 
-
<td>-</td><td>0.9</td><td>0.8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>-</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>-</td><td>5</td><td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>3</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0823-3.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1</td><td>2</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>3</td><td>10</td><td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>2</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td><td>12</td><td>12</td>
 
-
</tr>
 
-
</table>
 
-
:From left;
 
-
::#BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500] -> OK
 
-
::#BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]Single Digestion -> OK
 
-
::#BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]Double Digestion -> Bad
 
-
|}
 
-
 
-
==Week 2==
 
-
===24 August, 2008===
 
-
:(22/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)
 
-
::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)
 
-
::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/digestion|Digestion test]]'''
 
-
::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)
 
-
::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)
 
-
::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)
 
-
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>5</td><td>6</td><td>7</td><td>8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)</td>
 
-
<td>1</td><td>-</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)</td>
 
-
<td>-</td><td>1</td><td>-</td><td>-</td>
 
-
</tr><tr>
 
-
<td>BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)</td>
 
-
<td>-</td><td>-</td><td>1</td><td>-</td>
 
-
</tr><tr>
 
-
<td>BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)</td>
 
-
<td>-</td><td>-</td><td>-</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>EcoRⅠ</td>
 
-
<td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 4</td>
 
-
<td>0.9</td><td>0.9</td><td>0.9</td><td>0.9</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>8</td><td>8</td><td>8</td><td>8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0824-1.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1~8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td>
 
-
</tr>
 
-
</table>
 
-
:From left;
 
-
::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> ?
 
-
::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -> OK
 
-
::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) -> OK
 
-
::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) -> OK
 
-
::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK
 
-
::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -> OK
 
-
::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) -> OK
 
-
::#BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) -> None?
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0824-2.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>5~8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>9</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td></tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td>
 
-
</tr>
 
-
</table>
 
-
 
-
From the left;
 
-
（single digestuion）
 
-
::5, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK
 
-
::6, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006) -> OK
 
-
::7, BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007) -> 3400bp:Bad, 2200bp:OK
 
-
::8, BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006) -> OK
 
-
|}
 
-
 
-
===25 August, 2008===
 
-
:(24/8)--->'''[[Team:Chiba/protocol/digestion|Digestion]]'''
 
-
::#Double Digestion:BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
::#Double Digesiton:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
 
-
::#Single Digestion:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
 
-
::#BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1</td><td>2</td><td>3</td><td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>5</td><td>5</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>EcoRⅠ</td>
 
-
<td>0.5</td><td>o.5</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SpeⅠ</td>
 
-
<td>0.5</td><td>0.5</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 1</td>
 
-
<td>1</td><td>1</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 3</td>
 
-
<td>-</td><td>-</td><td>1</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>1</td><td>1</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>2</td><td>2</td><td>7.5</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td><td>10</td><td>1</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:--->[[Team:Chiba/protocol/gelcheck|Gel Check]]
 
-
{|align="justify"
 
-
|[[Image:Chiba-0825-1.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1~3</td><td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>10</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>-</td><td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td><td>6</td>
 
-
</tr>
 
-
</table>
 
-
:From right;
 
-
::#Double Digestion:BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK
 
-
::#Double Digesiton:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> ?(low)
 
-
::#Single Digestion:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> ?(low)
 
-
::#BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> OK(low)
 
-
|}
 
-
 
-
 
-
--->[[Team:Chiba/protocol/digestion|Digestion]]
 
-
(Double Digestion)
 
-
::#BBa_C0178[http://partsregistry.org/Part:BBa_C0178]
 
-
::#BBa_C0170[http://partsregistry.org/Part:BBa_C0170]
 
-
::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1</td><td>2</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>33ng/μl×60μl</td><td>33ng/μl×60μl</td><td>100ng/μl×20μl</td>
 
-
</tr>
 
-
<tr>
 
-
<td>PstⅠ</td>
 
-
<td>2</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>XbaⅠ</td>
 
-
<td>2</td><td>2</td><td>-</td>
 
-
</tr>]
 
-
<td>SpeⅠ</td>
 
-
<td>-</td><td>-</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 2</td>
 
-
<td>-</td><td>-</td><td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 3</td>
 
-
<td>10</td><td>10</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>10</td><td>10</td><td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>16</td><td>16</td><td>16</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>100</td><td>100</td><td>52</td>
 
-
</tr>
 
-
</table>
 
-
 
-
===26 August, 2008===
 
-
:(8/25)--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0825-2.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1~3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>30</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>6</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>36</td>
 
-
</tr>
 
-
</table>
 
-
::1, BBa_C0178[http://partsregistry.org/Part:BBa_C0178]
 
-
:::Insert:609bp -> OK
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0825-3.JPG]]
 
-
|
 
-
::2, BBa_C0170[http://partsregistry.org/Part:BBa_C0170]
 
-
:::Insert:609bp -> OK
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0825-4.JPG]]
 
-
|
 
-
::3, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
::: -> OK
 
-
|}
 
-
 
-
 
-
--->'''[[Team:Chiba/protocol/ligation/gelex|Gel Extract]]'''
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo Clean]]'''
 
-
:::#5μl
 
-
:::#5μl
 
-
:::#27μl
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0825-5.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1~3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td></tr>
 
-
<tr>
 
-
<td>H2O</td>
 
-
<td>8</td></tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td></tr>
 
-
</table>
 
-
 
-
:From left;
 
-
::#insert:C0178 -> OK
 
-
::#insert:C0170 -> OK?
 
-
::#vector:J04500 -> OK
 
-
|}
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/ligation/dephosphorylation|SAP]]'''
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>26</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SAP</td>
 
-
<td>1</td></tr>
 
-
<tr>
 
-
<td>SAP Buffer</td>
 
-
<td>3</td></tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>30μL</td></tr>
 
-
</table>
 
-
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo Clean]]'''
 
-
:::Sample No.3->9μl
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0826.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No.3</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td></tr>
 
-
</table>
 
-
::Sample No.3 -> OK ･･･30ng?
 
-
|}
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/ligation/ligation|Ligation]]'''
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>(1)</td><td>(2)</td><td>(3)</td><td>(4)</td><td>(5)</td>
 
-
</tr>
 
-
<tr>
 
-
<td>insert①C0178</td>
 
-
<td>1</td><td>-</td><td>-</td><td>1</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>insert②C0170</td>
 
-
<td>-</td><td>1</td><td>-</td><td>-</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>vector③J04500</td>
 
-
<td>-</td><td>-</td><td>2</td><td>2</td><td>2</td>
 
-
</tr>]
 
-
<td>ligase</td>
 
-
<td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer</td>
 
-
<td>2</td><td>2</td><td>2</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>6</td><td>6</td><td>5</td><td>4</td><td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td><td>10</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
::#insert①C0178
 
-
::#insert②C0170
 
-
::#vector③J04500
 
-
::#insert①C0178+vector③J04500
 
-
::#insert②C0170+vector③J04500
 
-
 
-
 
-
 
-
===27 August, 2008===
 
-
[[Team:Chiba/protocol/digestion|Digestion Test]]
 
-
::Sample DNA : BBa_R0010[http://partsregistry.org/Part:BBa_R0010]
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1</td><td>2</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>PstⅠ</td>
 
-
<td>-</td><td>-</td><td>0.5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>EcoRⅠ</td>
 
-
<td>-</td><td>0.5</td><td>0.5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 3</td>
 
-
<td>-</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>-</td><td>-</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>-</td><td>7.5</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>1</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0827.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1</td><td>2</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td><td>5</td><td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>H2O</td>
 
-
<td>4</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td><td>6</td><td>6</td>
 
-
</tr>
 
-
</table>
 
-
:From left;<br>
 
-
:#BBa_R0010
 
-
:#Single Digestion
 
-
:#Double Digestion
 
-
|}
 
-
 
-
 
-
 
-
:(26/8)--->'''[[Team:Chiba/protocol/PCR|Colony-PCR]]'''
 
-
::Colony PCR of 5 colonies from ligation plates (26/8-(4),(5)) and one from control plate(BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007)).
 
-
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">DNA Template</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dNTP mix</td>
 
-
<td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Foward Primer</td>
 
-
<td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Reverse Primer</td>
 
-
<td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>DNA polymerase</td>
 
-
<td>0.5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Thermopol Buffer</td>
 
-
<td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>28.5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>50μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
:95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃
 
-
 
-
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0827-2.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td></tr>
 
-
</table>
 
-
:From left;<br>
 
-
:1~5  insert:C0170 + vector:J04500  -> OK
 
-
:6    F2620･･Positive Control  -> OK
 
-
:7~11 insert:C0178 + vector:J04500 -> OK
 
-
|}
 
-
 
-
 
-
:--->(28/8)'''[[Team:Chiba/protocol/DNA Purification/sigma|Miniprep]]'''
 
-
 
-
 
-
 
-
'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
:competent cells : XL10G
 
-
::*BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
 
-
::*BBa_C0062(2006) [http://partsregistry.org/Part:BBa_C0062] -> no colony on plates
 
-
::*BBa_C0179(2007) [http://partsregistry.org/Part:BBa_C0179]
 
-
::*BBa_C0179(2006) [http://partsregistry.org/Part:BBa_C0179]
 
-
::*BBa_S03156(2007) [http://partsregistry.org/Part:BBa_S03156]
 
-
::*BBa_S03156(2006) [http://partsregistry.org/Part:BBa_S03156]
 
-
::*BBa_S03158(2007) [http://partsregistry.org/Part:BBa_S03158]
 
-
::*BBa_S03158(2006) [http://partsregistry.org/Part:BBa_S03158]
 
-
::*BBa_S03160(2007) [http://partsregistry.org/Part:BBa_S03160]
 
-
::*BBa_S0316(2006) [http://partsregistry.org/Part:BBa_S03160]
 
-
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
 
-
 
-
:--->(29/8)'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
 
-
 
-
 
-
===28 August, 2008===
 
-
:(27/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]
 
-
::#insert:C0170 + vector:J04500
 
-
::#insert:C0178 + vector:J04500
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/digestion|Digestion Test]]'''
 
-
::#insert:C0170 + vector:J04500
 
-
::#insert:C0178 + vector:J04500
 
-
 
-
<table width="500" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>3</td><td>4</td><td>5</td><td>6</td>
 
-
</tr>
 
-
<tr>
 
-
<td>insert:C0170 + vector:J04500 </td>
 
-
<td>1</td><td>-</td><td>1</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>insert:C0178 + vector:J04500 </td>
 
-
<td>-</td><td>1</td><td>-</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>EcoRⅠ</td>
 
-
<td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SpeⅠ</td>
 
-
<td>-</td><td>-</td><td>0.1</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 3</td>
 
-
<td>1</td><td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>-</td><td>-</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7.8</td><td>7.8</td><td>6.8</td><td>6.8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
 
-
:--->(29/8)'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0828.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1,2</td><td>3~6</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td><td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>H2O</td>
 
-
<td>4</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td><td>12</td>
 
-
</tr>
 
-
</table>
 
-
:From left;
 
-
::#　-> OK
 
-
::#　-> OK
 
-
::#　-> OK
 
-
::#　-> OK
 
-
::#　-> OK
 
-
::#　-> OK
 
-
|}
 
-
 
-
===29 August, 2008===
 
-
:--->'''[[Team:Chiba/protocol/digestion|Digestion Test]]'''
 
-
::#BBa_R0010(2007) [http://partsregistry.org/Part:BBa_R0010]
 
-
::#BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
 
-
::#BBa_C0179(2007) [http://partsregistry.org/Part:BBa_C0179]
 
-
::#BBa_C0179(2006) [http://partsregistry.org/Part:BBa_C0179]
 
-
::#BBa_S03156(2007) [http://partsregistry.org/Part:BBa_S03156]
 
-
::#BBa_S03156(2006) [http://partsregistry.org/Part:BBa_S03156]
 
-
::#BBa_S03158(2007) [http://partsregistry.org/Part:BBa_S03158]
 
-
::#BBa_S03158(2006) [http://partsregistry.org/Part:BBa_S03158]
 
-
::#BBa_S03160(2007) [http://partsregistry.org/Part:BBa_S03160]
 
-
::#BBa_S03160(2006) [http://partsregistry.org/Part:BBa_S03160]
 
-
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>S1~10</td><td>W1</td><td>W2~10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td><td>4</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>PstⅠ</td>
 
-
<td>-</td><td>0.075</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>EcoRⅠ</td>
 
-
<td>0.1</td><td>0.075</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 3</td>
 
-
<td>1</td><td>0.75</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>-</td><td>0.75</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7.9</td><td>4.35</td><td>5.8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0829.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No.</td>
 
-
<td>1~10</td><td>S1~S10,W1~W10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td><td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td><td>12</td></tr>
 
-
</table>
 
-
:From left;<br>
 
-
::1~5,S1,S5,W1~W5
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0829-2.JPG]]
 
-
|
 
-
:From left;<br>
 
-
::6~10,S6~S10,W6~W10
 
-
|}
 
-
 
-
 
-
 
-
:(27/8)--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0829-3.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>0.5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>4.5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6μL</td></tr>
 
-
</table>
 
-
 
-
::R0010 -> OK
 
-
|}
 
-
 
-
 
-
'''[[Team:Chiba/protocol/digestion|Digestion]]'''
 
-
::#BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::#BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
 
-
::#BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
 
-
::#BBa_R0010(2006) [http://partsregistry.org/Part:BBa_R0010]
 
-
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1~3</td><td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>100ng/μL×20</td><td>200ng/μL×30</td>
 
-
</tr>
 
-
<tr>
 
-
<td>PstⅠ</td>
 
-
<td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>XbaⅠ</td>
 
-
<td>2</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SpeⅠ</td>
 
-
<td>-</td><td>2</td></tr>
 
-
<tr>
 
-
<td>Buffer 2</td>
 
-
<td>-</td><td>5</td></tr>
 
-
<tr>
 
-
<tr>
 
-
<td>Buffer 3</td>
 
-
<td>5</td><td>-</td></tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>5</td><td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>16</td><td>6</td></tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>50</td><td>50</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0829-4.JPG]]
 
-
|
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No.</td>
 
-
<td>1~3</td><td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>10</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>-</td><td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td><td>12</td></tr>
 
-
</table>
 
-
 
-
:insert-I9026 : 685bp -> None<br>
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0829-5.JPG]]
 
-
|
 
-
:insert-I9030 : 745bp -> None<br>
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0829-6.JPG]]
 
-
|
 
-
:insert-S03154 : 685bp -> None<br>
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0829-7.JPG]]
 
-
|
 
-
:vector-R0010 : 2079bp -> OK<br>
 
-
|}
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/ligation/gelex|Gel Extract]]'''
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo Clean]]'''
 
-
:::->15μL
 
-
 
-
:--->'''[[Team:Chiba/protocol/ligation/dephosphorylation|SAP]]'''
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>15</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SAP</td>
 
-
<td>2</td></tr>
 
-
<tr>
 
-
<td>SAP Buffer</td>
 
-
<td>3</td></tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>10</td></tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>30μL</td></tr>
 
-
</table>
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo Clean]]'''
 
-
 
-
::--->Suspension! (We got information, by the team-output, to the effect that the density of the sample is too low.)
 
-
 
-
 
-
===30 August, 2008===
 
-
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
:competent cells : XL10G
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_S03156(2007) [http://partsregistry.org/Part:BBa_S03156]
 
-
::*BBa_S03158(2007) [http://partsregistry.org/Part:BBa_S03158]
 
-
::*BBa_S03160(2007) [http://partsregistry.org/Part:BBa_S03160]
 
-
::*BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
 
-
::*BBa_C0179(2007) [http://partsregistry.org/Part:BBa_C0179]
 
-
 
-
:--->(31/8)'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
 
-
 
-
 
-
:(29/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
::#insert:C0170 + vector:J04500
 
-
::#insert:C0178 + vector:J04500
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/digestion|Digestion Test]]'''
 
-
::*insert:C0170 + vector:J04500 -> Sample No.1~4
 
-
::*insert:C0178 + vector:J04500 -> Sample No.5~8
 
-
::*BBa_T9002(2007)[http://partsregistry.org/Part:BBa_T9002] -> Sample No.9
 
-
::*Single Digestion of Sample No.1~9 -> Sample No.10~18
 
-
::*Double Digestion of Sample No.1~9 -> Sample No.19~27
 
-
 
-
:<table width="500" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="270">Sample No.</td><td>Single : 10~18</td><td>Double : 19~27</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td><td>1</td><td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>XbaⅠ</td><td>0.1</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SpeⅠ</td><td>-</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 2</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td><td>6.9</td><td>2.8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0830.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1~9</td><td>10~18</td><td>19~27</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td><td>10</td><td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>4</td><td>-</td><td>-</td></tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td><td>12</td><td>12</td>
 
-
</tr>
 
-
</table>
 
-
:From left;<br>
 
-
::Sanple No.1~13
 
-
::-> OK
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0830-2.JPG]]
 
-
|
 
-
:From left;<br>
 
-
::Sample No.14~17,24~16
 
-
::14 -> OK
 
-
::15,16 -> Bad
 
-
::17 -> None
 
-
::24~16 -> Bad
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0830-3.JPG]]
 
-
|
 
-
:From left;<br>
 
-
::Sample No.18,27,19~23
 
-
::18,27 -> Bad
 
-
::19~22 -> OK
 
-
::23 -> OK??
 
-
|}
 
-
 
-
==Week 3==
 
-
===31 August, 2008===
 
-
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
:Competent Cells : XL10G
 
-
::*Plac+RBS+LasI
 
-
::*Plac+RBS+RhlI
 
-
 
-
'''[[Team:Chiba/protocol/digestion|Digestion]]'''
 
-
::#BBa_I9026[http://partsregistry.org/Part:BBa_I9026](2007)
 
-
::#BBa_I9030[http://partsregistry.org/Part:BBa_I9030](2006)
 
-
::#BBa_S03154[http://partsregistry.org/Part:BBa_S03154](2007)
 
-
::#BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1~3</td><td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td><td>12</td><td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>PstⅠ</td>
 
-
<td>0.2</td><td>0.2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>XbaⅠ</td><td>0.2</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SpeⅠ</td><td>-</td><td>0.4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 2</td>
 
-
<td>-</td><td>1.5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 3</td>
 
-
<td>2</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>2</td><td>1.5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>3.6</td><td>1.4</td></tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>20</td><td>15</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
:--->(1/9)'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
 
-
 
-
:(30/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/digestion|Digestion test]]'''
 
-
:*Plac+RBS+RhlI Sample No.2~5
 
-
:*BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
 
-
 
-
<table width="600" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample</td>
 
-
<td>Single Digesiton 2~5</td><td>Double Digestion 2~5</td><td>Single Digestion T9002①</td><td>Single Digesiton T9002②</td><td><Double Digestion T9002①②</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td><td>1</td><td>3</td><td>3</td><td>1</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>XbaⅠ</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SpeⅠ</td><td>-</td><td>0.1</td><td>-</td><td>-</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 2</td>
 
-
<td>0.9</td><td>0.8</td><td>0.9</td><td>0.9</td><td>0.8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td><td>5</td><td>5</td><td>7</td><td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td><td>10</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0831-p87.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>Plac+RBS+RhlI Sample No.2~5, BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①②
 
-
</td><td>Single & Double Digestion</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td><td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td><td>2</td></tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>4</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td><td>12</td>
 
-
</tr>
 
-
</table>
 
-
:From Left
 
-
::*Single Digestion Plac+RBS+RhlI 2~5
 
-
::*Single Digestion BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
 
-
::*Double Digestion Plac+RBS+RhlI 2~5
 
-
::*Double Digestion BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
 
-
::*BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
 
-
|}
 
-
 
-
===1 September, 2008===
 
-
:(31/8)--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0901.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1~3</td><td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>20</td><td>15</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>4</td><td>3</td></tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>24</td><td>18</td>
 
-
</tr>
 
-
</table>
 
-
:From left;
 
-
::insert-1(I9026)
 
-
::insert-2(I9030)
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0901-2.JPG]]
 
-
|
 
-
::insert-3(S03154)
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0901-3.JPG]]
 
-
|
 
-
::vector-4(R0010)
 
-
|}
 
-
 
-
:--->zymo
 
-
::vector-4(R0010)
 
-
::-> 15μL
 
-
 
-
:--->SAP
 
-
::vectr-4(R0010)
 
-
 
-
:--->Zymo
 
-
::1 -> 7μL
 
-
::2 -> 7
 
-
::3 -> 7
 
-
::4 -> 20
 
-
 
-
 
-
 
-
'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
:Competent cells : BW ΔFliC  40μL
 
-
::*Plac+RBS+LasI : Sample No.L1~L4
 
-
::*Plac+RBS+RhlI : Sample No.R1~R4
 
-
 
-
:--->(2/9)OD
 
-
 
-
===2 September, 2008===
 
-
:(31/8)--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0902.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①</td><td>②</td><td>③</td><td>④</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>3</td><td>3</td><td>3</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td><td>2</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td><td>7</td><td>7</td><td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td><td>12</td><td>12</td><td>12</td>
 
-
</tr>
 
-
</table>
 
-
:From left;
 
-
::I9026
 
-
::I9030
 
-
::S03154
 
-
|}
 
-
 
-
 
-
 
-
:(1/9)--->'''[[Team:Chiba/protocol/PCR|Colony-PCR]]'''
 
-
::Colony PCR of 8 colonies from ligation plates (1/9-(1),(2)) and one from control plate(BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007)).
 
-
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">DNA Template</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dNTP mix</td>
 
-
<td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Foward Primer</td>
 
-
<td>0.3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Reverse Primer</td>
 
-
<td>0.3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>DNA polymerase TAQ</td>
 
-
<td>0.5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Thermopol Buffer</td>
 
-
<td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>20.5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>30μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
:95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃
 
-
 
-
 
-
--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0902-2.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>1</td></tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td>
 
-
</tr>
 
-
</table>
 
-
:From left;
 
-
:*Plac+RBS+RhlI+LVA
 
-
::R1 -> OK
 
-
::R2 -> Bad
 
-
::R3~R7 -> OK
 
-
::R8 -> Bad
 
-
|}
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0902-3.JPG]]
 
-
|
 
-
:From left;
 
-
:*Plac+RBS+CinI+LVA
 
-
::C1,C2 -> OK
 
-
::C3 -> Bad
 
-
::C4~C6 -> OK
 
-
|}
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0902-4.JPG]]
 
-
|
 
-
:From left;
 
-
:*Plac+RBS+CinI+LVA
 
-
::C7,C8 -> OK
 
-
:*BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007):Positive control -> OK
 
-
|}
 
-
 
-
 
-
:--->(3/9)'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
 
-
 
-
 
-
 
-
:(1/9)--->OD測
 
-
 
-
 
-
 
-
'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
:Competent cells : XL10G  30μL
 
-
::*C0161(2007)
 
-
::*C0161(2006)
 
-
::*C0261(2007)
 
-
::*C0261(2006)
 
-
 
-
:--->(4/9)miniprep
 
-
 
-
===3 September, 2008===
 
-
:(2/9)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0903.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td>
 
-
</tr>
 
-
</table>
 
-
:From left;
 
-
::*S03154 -> OK
 
-
::*R1,R3~R7  (Plac+RBS+RhlI+LVA) -> OK
 
-
|}
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0903-2.JPG]]
 
-
|
 
-
:From left;
 
-
::*C1,C2,C4~C8  (Plac+RBS+CinI+LVA) -> OK
 
-
|}
 
-
 
-
:--->'''[[Team:Chiba/protocol/digestion|Digestion test]]'''
 
-
::*R1,R3~R7  (Plac+RBS+RhlI+LVA)
 
-
::*C1,C2,C4~C8  (Plac+RBS+CinI+LVA)
 
-
 
-
<table width="500" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Digestion</td>
 
-
<td>Single</td><td>Double</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>XbaⅠ</td>
 
-
<td>0.1</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>PstⅠ</td>
 
-
<td>0.1</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 2</td>
 
-
<td>0.9</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 3</td>
 
-
<td>-</td><td>-0.8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td><td>5</td></tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0903-3.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td></tr>
 
-
<tr><tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td>
 
-
</tr>
 
-
</table>
 
-
:From left;
 
-
:*Single Digestion : R1,R3~R7 -> OK
 
-
:*Single Digestion : C1,C2,C4~C8 -> OK
 
-
|}
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0903-4.JPG]]
 
-
|
 
-
:From left;
 
-
:*Double Digestion : R1,R3~R7 -> OK
 
-
:*Double Digestion : C1,C2,C4~C8 -> OK
 
-
|}
 
-
 
-
 
-
(2/9)--->'''Phenotype-test : mix'''
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No.</td>
 
-
<td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td>6</td><td>7</td><td>8</td><td>9</td><td>10</td><td>11</td>
 
-
</tr>
 
-
<tr>
 
-
<td>L1</td>
 
-
<td>2</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<tr><td>L2</td><td>-</td><td>2</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr>
 
-
<tr><td>L3</td><td>-</td><td>-</td><td>2</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr>
 
-
<tr><td>L4</td><td>-</td><td>-</td><td>-</td><td>2</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr>
 
-
<tr><td>R1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr>
 
-
<tr><td>R2</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr>
 
-
<tr><td>R3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1</td><td>-</td><td>-</td><td>-</td><td>-</td></tr>
 
-
<tr><td>R4</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1</td><td>-</td><td>-</td><td>-</td></tr>
 
-
 
-
<tr><td>BBa_S03154[http://partsregistry.org/Part:BBa_S03154]</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>2</td><td>-</td><td>-</td></tr>
 
-
<tr><td>BBa_T9002[http://partsregistry.org/Part:BBa_T9002]</td><td>2</td><td>2</td><td>2</td><td>2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>2</td><td>1</td><td>1</td></tr>
 
-
<tr><td>AHL</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr>
 
-
</table>
 
-
:::|
 
-
:::| 8h
 
-
:::V
 
-
::Spindown
 
-
:::|
 
-
:::|
 
-
:::V
 
-
::the intensity GFP
 
-
 
-
===4 September, 2008===
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0904.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①</td><td>②</td><td>③</td><td>④</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>3</td><td>3</td><td>3</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td><td>2</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td><td>7</td><td>7</td><td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td><td>12</td><td>12</td><td>12</td>
 
-
</tr>
 
-
</table>
 
-
:From left;
 
-
:
 
-
:
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0904-2.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①</td><td>②</td><td>③</td><td>④</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>3</td><td>3</td><td>3</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td><td>2</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td><td>7</td><td>7</td><td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td><td>12</td><td>12</td><td>12</td>
 
-
</tr>
 
-
</table>
 
-
:From left;
 
-
:
 
-
:
 
-
|}
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0904-3.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①</td><td>②</td><td>③</td><td>④</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>3</td><td>3</td><td>3</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td><td>2</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td><td>7</td><td>7</td><td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td><td>12</td><td>12</td><td>12</td>
 
-
</tr>
 
-
</table>
 
-
:From left;
 
-
:
 
-
:
 
-
|}
 
-
 
-
===5 September, 2008===
 
-
 
-
===6 September, 2008===
 
-
 
-
==Week 4==
 
-
===7 September, 2008===
 
-
 
-
===8 September, 2008===
 
-
 
-
===9 September, 2008===
 
-
 
-
===10 September, 2008===
 
-
 
-
===11 September, 2008===
 
-
 
-
===12 September, 2008===
 
-
 
-
===13 September, 2008===
 
-
:'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
::#BBa_C0161[http://partsregistry.org/Part:BBa_Co161](2007)
 
-
::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
::#BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
 
-
{|align="justify"
 
-
|[[Image:Chiba-0913.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1~3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td></tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td></tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>4</td></tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td></tr>
 
-
</table>
 
-
:From left;
 
-
::
 
-
::
 
-
 
-
|}
 
-
 
-
 
-
 
-
--->'''[Team:Chiba/protocol/digestion|Digestion]]'''
 
-
::#BBa_C0161[http://partsregistry.org/Part:BBa_Co161](2007)
 
-
::#BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
::#BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
 
-
 
-
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①</td><td>②</td><td>③</td><td>④</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>5</td><td>5</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>EcoRⅠ</td>
 
-
<td>0.5</td><td>o.5</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SpeⅠ</td>
 
-
<td>0.5</td><td>0.5</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 1</td>
 
-
<td>1</td><td>1</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 3</td>
 
-
<td>-</td><td>-</td><td>1</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>1</td><td>1</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>2</td><td>2</td><td>7.5</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td><td>10</td><td>1</td>
 
-
</tr>
 
-
</table>
 
-
 
-
==Week 5==
 
-
===14 September, 2008===
 
-
:(13/9)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
 
-
 
-
 
-
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
:Competent cells : XL10G
 
-
::*BBa_T9002[http://partsregistry.org/Part:BBa_T9002](2007)
 
-
::*Plac+RhlI+LVA
 
-
::*Plac+CinI+LVA
 
-
 
-
:--->(15/9)
 
-
 
-
===15 September, 2008===
 
-
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
:competent cells : BW⊿FliC
 
-
::*BBa_S03623 [http://partsregistry.org/Part:BBa_S03623]
 
-
::*BBa_K084007 [http://partsregistry.org/Part:BBa_K084007]
 
-
::*BBa_K084008 [http://partsregistry.org/Part:BBa_K084008]
 
-
::*BBa_K084009 [http://partsregistry.org/Part:BBa_K084009]
 
-
::*BBa_T9002 [http://partsregistry.org/Part:BBa_T9002]
 
-
 
-
(15/9)->cultured in new LB-Amp medium. <Br>
 
-
added AHL(total concentration was 0nM,1nM,10nM,100nM,1000nM)<Br>
 
-
::-->check green fluorescence intensity(Excitation:485nm,Emission:527nm)
 
 +
===Cross-Talk Check===
 +
#LuxR vs LuxI('''OK'''),LasI('''OK'''),CinI('''No'''),RhlI('''OK''')
 +
#LuxI vs LuxR,LasR,CinR,RhlR-->発現を確認できず
-
Cultured transformants(LB,37℃,14h)<Br>
+
===time-delay test===
-
*BBa_K084007~BBa_K084009
+
[[Team:Chiba/protocol/phenotype/timedelay/j|time-delay protocol]]
-
*BBa_T9002
+
-
*BBa_S03623
+
-
cultured above in new LB medium.<Br>
+
-
-->mix(16/9)
+
-
===16 September, 2008===
+
==連絡==
-
(15/9)->mix cultures in the way prescribed in the Table below.
+
===pptに載せたいもの===
-
::-->check green fluorescence intensity(Excitation:485nm,Emission:527nm)
+
====作ったもの====
 +
*絵で、わかりやすく。～Isごとに色を変える。
 +
====time-delay実験結果の整理(25/9)====
 +
*実験操作を分かりやすく書く
 +
*比較するもの
 +
**LuxI,LasI,RhlI
 +
**TAG有/無
 +
**温度
 +
**菌数比
 +
**株
 +
*ポイント
 +
#正しく書きだす
 +
#推論を立てる（帰納的に、結論を急がないこと）
 +
====固体培地spot実験(28/9)====
 +
前日
 +
*IPTG入りLB-Agar-amp（20ml）に、T9002を2ml(10<sup>3</sup>-10<sup>5</sup> cells/2ml)撒く-->37℃,O/N
 +
*～Is（5種類）をLB-Amp,2ml培養
 +
当日
 +
*T9002がコロニーを形成したプレートの中心に、~Isの培養液を1μl,spotする。
 +
*gfpの発現が同心円状に広がっていく。その計時変化を記録する。
-
{| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"
+
{| style="color:white;" cellpadding="3" cellspacing="3" border="0" width="100%" align="center" class="menu" |
!align="center"|[[Team:Chiba/j|ホーム]]
!align="center"|[[Team:Chiba/j|ホーム]]
!align="center"|[[Team:Chiba/Team/j|メンバー紹介]]
!align="center"|[[Team:Chiba/Team/j|メンバー紹介]]
!align="center"|[[Team:Chiba/Project/j|プロジェクト紹介]]
!align="center"|[[Team:Chiba/Project/j|プロジェクト紹介]]
-
!align="center"|[[Team:Chiba/Parts/j|Parts Submitted to the Registry]]
+
!align="center"|[[Team:Chiba/Parts/j|作成したDNAパーツ]]
-
!align="center"|[[Team:Chiba/Modeling/j|モデリング]]
+
!align="center"|[[Team:Chiba/Internal|ノート]]
!align="center"|[[Team:Chiba/Internal|ノート]]
|}
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Latest revision as of 11:27, 14 December 2008

>ノートに戻る


Contents

送受信班の実験ノート

モジュール解説

感度差による応答時間の差

任意の順番と、delay-timeをもった遺伝子発現を実現するために、バクテリア間の細胞間通信、クオラムセンシングを利用する。クオラムセンシングでは、応答閾値を超えるAHLが蓄積されたとき初めて、目的遺伝子の発現がおこる。AHL情報がじゅうぶんゆっくり蓄積するとき、ひとつのAHL送信装置に対して、感度の異なる複数の受信装置によってさまざまな出力装置を独立に起動すれば、感度の高いAHL 受信機ほど先に起動する。

Controlling the Delay time of the AHL signaling

LuxIのつくるAHLに対して、感度差による応答時間差は、２つの方法で実現することができる。

  1. LuxR/ Pluxの感度変異体を用いる場合
    LuxRの変異体を用いれば、pluxがonになるAHL濃度が<1nMから~10nMまで変化する。[http://authors.library.caltech.edu/5553/ C. H. Collins.et al.:Mol.Microbiol.2005.55(3).712–723]
  2. クロストークを使う場合
AHL=Acyl-homoserine Lactoneには、そのアシル基が異なる種類がある。luxIが合成するAHLは、3-oxo-hyxanoyl-Homoserine Lactone (=OHHL)であり、3-oxo-dodecanoyl-homoserine Lactone(=ODHL)や、butyryl-homoserine Lactone(=HHL)が存在する。それぞれ、合成酵素はLasI、RhlI（ともにシュードモナス由来）と呼ばれる。このほかのAHL分子と、その合成酵素を以下に示した。これらのAHL分子は、アシル基のみが異なるため、一定の割合でLuxRと相互作用する（cross-talkする）。たとえば、OdHLとLuxRとのcross-talk（LasI-LuxR）は、OHHLとLuxRの場合（LuxI-LuxR）の1/20である。AHL分子の蓄積速度が一定ならば，luxI-LuxR/Pluxよりも20倍遅くスイッチが入ることとなる。
  • AHLsおよびその合成酵素
Strain Signal Molecule Enzyme
P.aeruginosa C4-HSL RhlI
V. fisheri C6-3-oxo-HSL LuxI
A.tumefaciens C8-3-oxo-HSL TraI
P.aeruginosa C10-3-oxo-HSL VanI
P.aeruginosa C12-3-oxo-HSL LasI
R.leguminosarum C14-3-hydroxy-HSL CinI

[http://partsregistry.org/Part:BBa_F2620:Specificity AHLsについて]

実験

Construction(From BioBrick)

  • Sender(Autoinducer synthase)
LuxI,LasI,CinI,RhlI(lacプロモーター)
  • Receiver
LuxR,LasR,CinR,RhlR


Cross-Talk Check

  1. LuxR vs LuxI(OK),LasI(OK),CinI(No),RhlI(OK)
  2. LuxI vs LuxR,LasR,CinR,RhlR-->発現を確認できず

time-delay test

time-delay protocol

連絡

pptに載せたいもの

作ったもの

  • 絵で、わかりやすく。～Isごとに色を変える。

time-delay実験結果の整理(25/9)

  • 実験操作を分かりやすく書く
  • 比較するもの
    • LuxI,LasI,RhlI
    • TAG有/無
    • 温度
    • 菌数比
    • 株
  • ポイント
  1. 正しく書きだす
  2. 推論を立てる（帰納的に、結論を急がないこと）

固体培地spot実験(28/9)

前日

  • IPTG入りLB-Agar-amp（20ml）に、T9002を2ml(103-105 cells/2ml)撒く-->37℃,O/N
  • ～Is（5種類）をLB-Amp,2ml培養

当日

  • T9002がコロニーを形成したプレートの中心に、~Isの培養液を1μl,spotする。
  • gfpの発現が同心円状に広がっていく。その計時変化を記録する。


ホーム メンバー紹介 プロジェクト紹介 作成したDNAパーツ ノート
Retrieved from "http://2008.igem.org/Team:Chiba/jk/%CE%B2"