Team:Chiba/jk/β/weak 3

From 2008.igem.org

(Difference between revisions)
Line 1: Line 1:
>[[Team:Chiba/jk/β|送受信班]]
>[[Team:Chiba/jk/β|送受信班]]
 +
 +
==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===

Revision as of 07:18, 20 September 2008

>送受信班

Contents

Week 3

31 August, 2008

Transformation
Competent Cells : XL10G
  • Plac+RBS+LasI
  • Plac+RBS+RhlI

Digestion

  1. BBa_I9026[http://partsregistry.org/Part:BBa_I9026](2007)
  2. BBa_I9030[http://partsregistry.org/Part:BBa_I9030](2006)
  3. BBa_S03154[http://partsregistry.org/Part:BBa_S03154](2007)
  4. BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
Sample No 1~34
Sample DNA1210
PstⅠ 0.20.2
XbaⅠ0.2-
SpeⅠ-0.4
Buffer 2 -1.5
Buffer 3 2-
BSA 21.5
dH2O 3.61.4
TOTAL 2015


--->(1/9)Gel Check


(30/8)--->Mini prep


--->Digestion test
  • Plac+RBS+RhlI Sample No.2~5
  • BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
Sample Single Digesiton 2~5Double Digestion 2~5Single Digestion T9002①Single Digesiton T9002②<Double Digestion T9002①②
Sample DNA13313
XbaⅠ0.10.10.10.10.1
SpeⅠ-0.1--0.1
Buffer 2 0.90.80.90.90.8
BSA 11111
dH2O 75575
TOTAL 1010101010


--->Gel Check

Chiba-0831-p87.JPG
Sample No Plac+RBS+RhlI Sample No.2~5, BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①② Single & Double Digestion
Sample DNA 110
Loading Dye 12
dH2O 4-
TOTAL 612
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)--->Gel Check
Chiba-0901.JPG
Sample No 1~34
Sample DNA 2015
Loading Dye 43
TOTAL 2418
From left;
insert-1(I9026)
insert-2(I9030)
Chiba-0901-2.JPG
insert-3(S03154)
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


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)--->Gel Check
Chiba-0902.JPG
Sample No ①②③④
Sample DNA 3333
Loading Dye 2222
dH2O 7777
TOTAL 12121212
From left;
I9026
I9030
S03154


(1/9)--->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)).
DNA Template 1
dNTP mix 5
Foward Primer 0.3
Reverse Primer 0.3
DNA polymerase TAQ 0.5
Thermopol Buffer 3
dH2O 20.5
TOTAL 30μL


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


--->Gel Check

Chiba-0902-2.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
  • Plac+RBS+RhlI+LVA
R1 -> OK
R2 -> Bad
R3~R7 -> OK
R8 -> Bad
Chiba-0902-3.JPG
From left;
  • Plac+RBS+CinI+LVA
C1,C2 -> OK
C3 -> Bad
C4~C6 -> OK
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)Mini prep



(1/9)--->OD測


Transformation

Competent cells : XL10G 30μL
  • C0161(2007)
  • C0161(2006)
  • C0261(2007)
  • C0261(2006)
--->(4/9)miniprep

3 September, 2008

(2/9)--->Mini prep
--->Gel Check
Chiba-0903.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
  • S03154 -> OK
  • R1,R3~R7 (Plac+RBS+RhlI+LVA) -> OK
Chiba-0903-2.JPG
From left;
  • C1,C2,C4~C8 (Plac+RBS+CinI+LVA) -> OK
--->Digestion test
  • R1,R3~R7 (Plac+RBS+RhlI+LVA)
  • C1,C2,C4~C8 (Plac+RBS+CinI+LVA)
Digestion SingleDouble
Sample DNA 13
XbaⅠ 0.10.1
PstⅠ 0.10.1
Buffer 2 0.9-
Buffer 3 --0.8
BSA 11
dH2O 75
TOTAL 1010


--->Gel Check
Chiba-0903-3.JPG
Sample DNA 10
Loading Dye 2
TOTAL 12
From left;
  • Single Digestion : R1,R3~R7 -> OK
  • Single Digestion : C1,C2,C4~C8 -> OK
Chiba-0903-4.JPG
From left;
  • Double Digestion : R1,R3~R7 -> OK
  • Double Digestion : C1,C2,C4~C8 -> OK


(2/9)--->Phenotype-test : mix

Sample No. 1234567891011
L1 2----------
L2-2---------
L3--2--------
L4---2-------
R1----1------
R2-----1-----
R3------1----
R4-------1---
BBa_S03154[http://partsregistry.org/Part:BBa_S03154]--------2--
BBa_T9002[http://partsregistry.org/Part:BBa_T9002]22221111211
AHL11111111111
|
| 8h
V
Spindown
|
|
V
the intensity GFP

4 September, 2008

--->Gel Check
Chiba-0904.JPG
Sample No ①②③④
Sample DNA 3333
Loading Dye 2222
dH2O 7777
TOTAL 12121212
From left;
Chiba-0904-2.JPG
Sample No ①②③④
Sample DNA 3333
Loading Dye 2222
dH2O 7777
TOTAL 12121212
From left;
Chiba-0904-3.JPG
Sample No ①②③④
Sample DNA 3333
Loading Dye 2222
dH2O 7777
TOTAL 12121212
From left;

5 September, 2008

6 September, 2008

ホーム メンバー紹介 プロジェクト紹介 Parts Submitted to the Registry モデリング ノート
Retrieved from "http://2008.igem.org/Team:Chiba/jk/%CE%B2/weak_3"