Team:Chiba/jk/β

From 2008.igem.org

(Difference between revisions)
Line 1,175: Line 1,175:
::#insert①C0178+vector③J04500
::#insert①C0178+vector③J04500
::#insert②C0170+vector③J04500
::#insert②C0170+vector③J04500
 +
 +
===27 August, 2008===
===27 August, 2008===
Line 1,253: Line 1,255:
::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)).
::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="500" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
<tr>
<tr>
-
<td width="257">Sample No</td>
+
<td width="257">DNA Template</td>
-
<td>(4)-1~5</td><td>(5)-1~5</td><td>(F2620)</td>
+
<td>1</td>
</tr>
</tr>
<tr>
<tr>
-
<td>insert①C0178</td>
+
<td>dNTP mix</td>
-
<td>1</td><td>-</td><td>-</td>
+
<td>5</td>
</tr>
</tr>
<tr>
<tr>
-
<td>insert②C0170</td>
+
<td>Foward Primer</td>
-
<td>-</td><td>1</td><td>-</td>
+
<td>5</td>
</tr>
</tr>
<tr>
<tr>
-
<td>vector③J04500</td>
+
<td>Reverse Primer</td>
-
<td>-</td><td>-</td><td>2</td>
+
<td>5</td>
-
</tr>]
+
-
<td>ligase</td>
+
-
<td>1</td><td>1</td><td>1</td>
+
</tr>
</tr>
<tr>
<tr>
-
<td>Buffer</td>
+
<td>DNA polymerase</td>
-
<td>2</td><td>2</td><td>2</td>
+
<td>0.5</td>
 +
</tr>
 +
<tr>
 +
<td>Thermopol Buffer</td>
 +
<td>5</td>
</tr>
</tr>
<tr>
<tr>
<td>dH2O</td>
<td>dH2O</td>
-
<td>6</td><td>6</td><td>5</td>
+
<td>28.5</td>
</tr>
</tr>
<tr>
<tr>
<td>TOTAL</td>
<td>TOTAL</td>
-
<td>10</td><td>10</td><td>10</td>
+
<td>50μL</td>
</tr>
</tr>
</table>
</table>
 +
 +
 +
:95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )・・・25cycles -> 72℃,10min -> 6℃
 +
Line 1,935: Line 1,942:
::vector-4(R0010)
::vector-4(R0010)
|}
|}
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/ligation/gelex|Gel Extract]]'''
 
-
 
:--->zymo
:--->zymo
::vector-4(R0010)
::vector-4(R0010)
::-> 15μL
::-> 15μL
-
 
:--->SAP
:--->SAP
Line 1,954: Line 1,956:
::4 -> 20
::4 -> 20
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+
===2 September, 2008===
 +
:(31/8)--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
{|align="justify"
{|align="justify"
-
|[[Image:Chiba-0901-5.JPG]]
+
|[[Image:Chiba-0902.JPG]]
|
|
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
<tr>
<tr>
<td width="257">Sample No</td>
<td width="257">Sample No</td>
-
<td>1~4</td>
+
<td>①</td><td>②</td><td>③</td><td>④</td>
</tr>
</tr>
<tr>
<tr>
<td>Sample DNA</td>
<td>Sample DNA</td>
-
<td>1</td></tr>
+
<td>3</td><td>3</td><td>3</td><td>3</td>
 +
</tr>
<tr>
<tr>
<td>Loading Dye</td>
<td>Loading Dye</td>
-
<td>1</td></tr>
+
<td>2</td><td>2</td><td>2</td><td>2</td>
 +
</tr>
<tr>
<tr>
<td>dH2O</td>
<td>dH2O</td>
-
<td>4</td>
+
<td>7</td><td>7</td><td>7</td><td>7</td>
</tr>
</tr>
<tr>
<tr>
<td>TOTAL</td>
<td>TOTAL</td>
-
<td>6μL</td>
+
<td>12</td><td>12</td><td>12</td><td>12</td>
</tr>
</tr>
</table>
</table>
:From left;
:From left;
-
::insert-1(I9026) -> OK
+
::I9026
-
::insert-2(I9030) -> OK
+
::I9030
-
::insert-3(S03154) -> None
+
::S03154
-
::vector-4(R0010) -> OK
+
|}
|}
-
:--->'''[[Team:Chiba/protocol/ligation/ligation|Ligation]]'''
+
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+
:(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>
<tr>
-
<td width="257">Sample No</td>
+
<td width="257">DNA Template</td>
-
<td>(1)</td><td>(2)</td><td>(3)</td><td>(4)</td><td>(5)</td>
+
<td>1</td>
</tr>
</tr>
<tr>
<tr>
-
<td>insert-1(I9026)</td>
+
<td>dNTP mix</td>
-
<td>3</td><td>-</td><td>3</td><td>-</td><td>-</td>
+
<td>5</td>
</tr>
</tr>
<tr>
<tr>
-
<td>insert-2(I9030)</td>
+
<td>Foward Primer</td>
-
<td>-</td><td>3</td><td>-</td><td>3</td><td>-</td>
+
<td>0.3</td>
</tr>
</tr>
<tr>
<tr>
-
<td>vector-4</td>
+
<td>Reverse Primer</td>
-
<td>3</td><td>3</td><td>-</td><td>-</td><td>3</td>
+
<td>0.3</td>
</tr>
</tr>
-
<td>ligase</td>
+
<tr>
-
<td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
+
<td>DNA polymerase TAQ</td>
 +
<td>0.5</td>
</tr>
</tr>
<tr>
<tr>
-
<td>Buffer</td>
+
<td>Thermopol Buffer</td>
-
<td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
+
<td>3</td>
</tr>
</tr>
<tr>
<tr>
<td>dH2O</td>
<td>dH2O</td>
-
<td>2</td><td>2</td><td>5</td><td>5</td><td>5</td>
+
<td>20.5</td>
</tr>
</tr>
<tr>
<tr>
<td>TOTAL</td>
<td>TOTAL</td>
-
<td>10</td><td>10</td><td>10</td><td>10</td><td>10</td>
+
<td>30μL</td>
</tr>
</tr>
</table>
</table>
 +
:95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )・・・25cycles -> 72℃,10min -> 6℃
-
:--->:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
::#insert-1 + vector-4 (Plac+RhlI+LVA)
 
-
::#insert-2 + vector-4 (Plac+CinI+LVA)
 
-
::#insert-1 (Back-ground)
 
-
::#insert-2 (Back-ground)
 
-
::#vector-4 (Back-ground)
 
-
===2 September, 2008===
+
--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
-
:'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+
{|align="justify"
{|align="justify"
-
|[[Image:Chiba-0902.JPG]]
+
|[[Image:Chiba-0902-2.JPG]]
|
|
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
<tr>
<tr>
-
<td width="257">Sample No</td>
+
<td width="257">Sample DNA</td>
-
<td>1~3</td>
+
<td>1</td></tr>
-
</tr>
+
<tr>
<tr>
-
<td>Sample DNA</td>
+
<td>Loading Dye</td>
<td>1</td>
<td>1</td>
</tr>
</tr>
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td></tr>
 
<tr>
<tr>
<td>dH2O</td>
<td>dH2O</td>
Line 2,059: Line 2,057:
</table>
</table>
:From left;
:From left;
-
:
+
:*Plac+RBS+RhlI+LVA
-
:
+
::R1 -> OK
 +
::R2 -> Bad
 +
::R3~R7 -> OK
 +
::R8 -> Bad
|}
|}
-
 
-
'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
{|align="justify"
{|align="justify"
-
|[[Image:Chiba-0902-2.JPG]]
+
|[[Image:Chiba-0902-3.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;
:From left;
-
::
+
:*Plac+RBS+CinI+LVA
 +
::C1,C2 -> OK
 +
::C3 -> Bad
 +
::C4~C6 -> OK
|}
|}
-
 
-
'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
{|align="justify"
{|align="justify"
-
|[[Image:Chiba-0902-3.JPG]]
+
|[[Image:Chiba-0902-4.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;
:From left;
-
:
+
:*Plac+RBS+CinI+LVA
-
:
+
::C7,C8 -> OK
 +
:*BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007):Positive control -> OK
|}
|}
-
'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
+
:--->(3/9)'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
-
|[[Image:Chiba-0902-4.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;
+
-
:
+
-
|}
+
 
 +
 
 +
:(1/9)--->'''[[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
===3 September, 2008===
===3 September, 2008===
-
:'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
+
:(2/9)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 +
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
{|align="justify"
{|align="justify"
|[[Image:Chiba-0903.JPG]]
|[[Image:Chiba-0903.JPG]]
Line 2,167: Line 2,105:
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
<tr>
<tr>
-
<td width="257">Sample No</td>
+
<td width="257">Sample DNA</td>
-
<td>①</td><td>②</td><td>③</td><td>④</td>
+
<td>1</td>
-
</tr>
+
-
<tr>
+
-
<td>Sample DNA</td>
+
-
<td>3</td><td>3</td><td>3</td><td>3</td>
+
</tr>
</tr>
<tr>
<tr>
<td>Loading Dye</td>
<td>Loading Dye</td>
-
<td>2</td><td>2</td><td>2</td><td>2</td>
+
<td>1</td>
</tr>
</tr>
<tr>
<tr>
<td>dH2O</td>
<td>dH2O</td>
-
<td>7</td><td>7</td><td>7</td><td>7</td>
+
<td>4</td>
</tr>
</tr>
<tr>
<tr>
<td>TOTAL</td>
<td>TOTAL</td>
-
<td>12</td><td>12</td><td>12</td><td>12</td>
+
<td>6</td>
</tr>
</tr>
</table>
</table>
:From left;
:From left;
-
:
+
::*S03154 -> OK
-
:
+
::*R1,R3~R7  (Plac+RBS+RhlI+LVA) -> OK
-
 
+
|}
|}
Line 2,196: Line 2,129:
|[[Image:Chiba-0903-2.JPG]]
|[[Image:Chiba-0903-2.JPG]]
|
|
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+
: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>
<tr>
-
<td width="257">Sample No</td>
+
<td width="257">Digestion</td>
-
<td></td><td>②</td><td>③</td><td>④</td>
+
<td>Single</td><td>Double</td>
</tr>
</tr>
<tr>
<tr>
<td>Sample DNA</td>
<td>Sample DNA</td>
-
<td>3</td><td>3</td><td>3</td><td>3</td>
+
<td>1</td><td>3</td>
</tr>
</tr>
<tr>
<tr>
-
<td>Loading Dye</td>
+
<td>XbaⅠ</td>
-
<td>2</td><td>2</td><td>2</td><td>2</td>
+
<td>0.1</td><td>0.1</td>
</tr>
</tr>
<tr>
<tr>
-
<td>dH2O</td>
+
<td>PstⅠ</td>
-
<td>7</td><td>7</td><td>7</td><td>7</td>
+
<td>0.1</td><td>0.1</td>
</tr>
</tr>
<tr>
<tr>
-
<td>TOTAL</td>
+
<td>Buffer 2</td>
-
<td>12</td><td>12</td><td>12</td><td>12</td>
+
<td>0.9</td><td>-</td>
</tr>
</tr>
-
</table>
 
-
:From left;
 
-
:
 
-
:
 
-
 
-
|}
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0903-3.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
<tr>
<tr>
-
<td width="257">Sample No</td>
+
<td>Buffer 3</td>
-
<td></td><td>②</td><td>③</td><td>④</td>
+
<td>-</td><td>-0.8</td>
</tr>
</tr>
<tr>
<tr>
-
<td>Sample DNA</td>
+
<td>BSA</td>
-
<td>3</td><td>3</td><td>3</td><td>3</td>
+
<td>1</td><td>1</td>
-
</tr>
+
-
<tr>
+
-
<td>Loading Dye</td>
+
-
<td>2</td><td>2</td><td>2</td><td>2</td>
+
</tr>
</tr>
<tr>
<tr>
<td>dH2O</td>
<td>dH2O</td>
-
<td>7</td><td>7</td><td>7</td><td>7</td>
+
<td>7</td><td>5</td></tr>
-
</tr>
+
<tr>
<tr>
<td>TOTAL</td>
<td>TOTAL</td>
-
<td>12</td><td>12</td><td>12</td><td>12</td>
+
<td>10</td><td>10</td>
</tr>
</tr>
</table>
</table>
-
:From left;
 
-
:
 
-
:
 
-
|}
+
 
{|align="justify"
{|align="justify"
-
|[[Image:Chiba-0903-4.JPG]]
+
|[[Image:Chiba-0903-3.JPG]]
|
|
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
<tr>
<tr>
-
<td width="257">Sample No</td>
+
<td width="257">Sample DNA</td>
-
<td>①</td><td>②</td><td>③</td><td>④</td>
+
<td>10</td>
-
</tr>
+
-
<tr>
+
-
<td>Sample DNA</td>
+
-
<td>3</td><td>3</td><td>3</td><td>3</td>
+
</tr>
</tr>
<tr>
<tr>
<td>Loading Dye</td>
<td>Loading Dye</td>
-
<td>2</td><td>2</td><td>2</td><td>2</td>
+
<td>2</td></tr>
-
</tr>
+
<tr><tr>
-
<tr>
+
-
<td>dH2O</td>
+
-
<td>7</td><td>7</td><td>7</td><td>7</td>
+
-
</tr>
+
-
<tr>
+
<td>TOTAL</td>
<td>TOTAL</td>
-
<td>12</td><td>12</td><td>12</td><td>12</td>
+
<td>12</td>
</tr>
</tr>
</table>
</table>
:From left;
: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
|}
|}

Revision as of 15:18, 14 September 2008

β班の実験ノート

Contents

Week 1

17 August, 2008

Transformation
competent cells : XL10G
  • BBa_R0040(2008) [1] -> No colonies on plates
  • BBa_C0070(2008) [2] -> No colonies on plates
  • BBa_C0076(2008) [3] -> No colonies on plates
  • BBa_C0077(2008) [4] -> No colonies on plates
  • BBa_C0078(2008) [5] -> No colonies on plates
  • BBa_C0079(2008) [6] -> No colonies on plates

18 August, 2008

PCR
  • BBa_C0070(2008) [7]
  • BBa_C0076(2008) [8]
  • BBa_C0077(2008) [9]
  • BBa_C0078(2008) [10]
  • BBa_C0079(2008) [11]
  • BBa_F2620(2007) [12]・・・control


DNA Template 1
dNTP mix 10
Foward Primer 5
Reverse Primer 5
DNA polymerase 1
Thermopol Buffer 10
dH2O 68
TOTAL 100μL


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


--->Gel Check


Chiba-0818.JPG
Sample DNA 3
Loading Dye 2
dH2O 7
TOTAL 12μL
From left;
  • BBa_C0070(2008) [13] ->None
  • BBa_C0076(2008) [14] ->None
  • BBa_C0077(2008) [15] ->None
  • BBa_C0078(2008) [16] ->None
  • BBa_C0079(2008) [17] ->None
  • BBa_F2620(2007) [18]・・・control ->OK



Transformation

competent cells : XL10G

  • BBa_C0070(2007) [19]
  • BBa_C0070(2006) [20]
  • BBa_C0076(2007) [21]
  • BBa_C0076(2006) [22] -> No colonies on plates(19/8)
  • BBa_C0078(2007) [23]
  • BBa_C0078(2006) [24]
  • BBa_C0170(2007) [25]
  • BBa_C0170(2006) [26]
  • BBa_C0178(2007) [27]
  • BBa_C0178(2006) [28]


--->(20/8)Mini prep

19 August, 2008

PCR
  • BBa_I9026(2007) [29]
  • BBa_I9026(2006) [30]
  • BBa_I9030(2007) [31]
  • BBa_I9030(2006) [32]
  • BBa_S3154(2007) [33]
  • BBa_S3154(2006) [34]
  • BBa_F2620(2007) [35]・・・control


DNA Template 1
dNTP mix 10
Foward Primer 5
Reverse Primer 5
DNA polymerase VENT 1
Thermopol Buffer 5
dH2O 28
TOTAL 50μL


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


--->Gel Check
Chiba-0819.JPG
Sample DNA 3
Loading Dye 2
dH2O 7
TOTAL 12μL
From left;
  • BBa_I9026(2007) [36] ->None
  • BBa_I9026(2006) [37] ->None
  • BBa_I9030(2007) [38] ->None
  • BBa_I9030(2006) [39] ->None
  • BBa_S3154(2007) [40] ->None
  • BBa_S3154(2006) [41] ->None
  • BBa_F2620(2007) [42]・・・control ->OK


20 August, 2008

(18/8)--->Mini prep
  • BBa_C0070(2007) [43]
  • BBa_C0070(2006) [44]
  • BBa_C0076(2007) [45]
  • BBa_C0078(2007) [46]
  • BBa_C0078(2006) [47]
  • BBa_C0170(2007) [48]
  • BBa_C0170(2006) [49]
  • BBa_C0178(2007) [50]
  • BBa_C0178(2006) [51]


--->Gel Check
Chiba-0820-3.JPG
Sample DNA 3
Loading Dye 2
dH2O 7
TOTAL 12μL
From left;
  • BBa_C0076(2007) [52] -> None
  • BBa_C0078(2007) [53] -> None
  • BBa_C0078(2006) [54] -> None


Chiba-0820-4.JPG
Sample DNA 10
Loading Dye 4
dH2O 10
TOTAL 24μL
From left;
  • BBa_C0078(2007) [55] -> None
  • BBa_C0078(2006) [56] -> OK
  • BBa_C0076(2007) [57] -> None
  • BBa_C0070(2007) [58] -> OK
  • BBa_C0070(2006) [59] -> OK



Chiba-0820.JPG
Sample DNA 3
Loading Dye 2
dH2O 7
TOTAL 12μL
From left;
  • BBa_C0170(2007) [60] -> OK
  • BBa_C0170(2006) [61] -> OK
  • BBa_C0178(2007) [62] -> OK
  • BBa_C0178(2006) [63] -> OK


--->Digestion test

  • BBa_C0170(2007) [64]
  • BBa_C0170(2006) [65]
  • BBa_C0178(2007) [66]
  • BBa_C0178(2006) [67]
Sample No 12345678
BBa_C0170(2006) [68] 3---3---
BBa_C0170(2007) [69] -3---3--
BBa_C0178(2006) [70] --3---3-
BBa_C0178(2007) [71] ---3---3
EcoRⅠ 0.10.10.10.10.10.10.10.1
PstⅠ ----0.10.10.10.1
Buffer EcoRⅠ 11111111
BSA 11111111
dH2O 4.94.94.94.94.84.84.84.8
TOTAL 1010101010101010


--->Gel Check
Chiba-0820-2.JPG
Sample DNA 3
Loading Dye 2
dH2O 7
TOTAL 12μL
From left;
(Single Digestion 1~4)
  • BBa_C0170(2006) [72] -> OK
  • BBa_C0170(2007) [73] -> OK
  • BBa_C0178(2006) [74] -> OK
  • BBa_C0178(2007) [75] -> OK
(Double Digestion 1~4)
  • BBa_C0170(2006) [76] -> OK
  • BBa_C0170(2007) [77] -> OK
  • BBa_C0178(2006) [78] -> OK
  • BBa_C0178(2007) [79] -> OK

21 August, 2008

(20/8)--->PCR
  • BBa_C0070(2007) [80]
  • BBa_C0070(2006) [81]
  • BBa_C0076(2007) [82]
  • BBa_C0078(2007) [83]
  • BBa_C0078(2006) [84]


DNA Template 1
dNTP mix 10
Foward Primer 5
Reverse Primer 5
DNA polymerase TAQ 1
Thermopol Buffer 5
dH2O 28
TOTAL 50μL


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


--->Gel Check
Chiba-0821.JPG
Sample DNA 3
Loading Dye 2
dH2O 7
TOTAL 12μL
From left;
  • BBa_C0078(2007) [85] -> None
  • BBa_C0078(2006) [86] -> None
  • BBa_C0076(2007) [87] -> None
  • BBa_C0070(2007) [88] -> OK
  • BBa_C0070(2006) [89] -> OK


Transformation
competent cells : XL10G
  • BBa_S03154(2007) [90]
  • BBa_S03154(2006) [91]
  • BBa_I9026(2007) [92]
  • BBa_I9026(2006) [93]
  • BBa_I9030(2007) [94]
  • BBa_I9030(2006) [95]



--->(23/8)Digestion


22 August, 2008

Transformation
competent cells : XL10G
  • BBa_J13002(2007) [96]
  • BBa_J13002(2006) [97]
  • BBa_J04500(2007) [98]
  • BBa_J04500(2006) [99]
(Locate & 2 Punch ~iyama ver.~)
  • BBa_C0076(2008) [100] -> No colonies on plates
  • BBa_C0077(2008) [101] -> No colonies on plates
  • BBa_C0078(2008) [102] -> No colonies on plates
  • BBa_J04500(2008) [103] -> No colonies on plates


--->(24/8)Mini prep

23 August, 2008

(21/8)--->Mini prep


--->Digestion test


Sample No 1 2 3 4 5 6 7 8 9101112131415161718192021
BBa_I9030(2007) [117] 3------3------3------
BBa_I9030(2006) [118] -3------3------3-----
BBa_S03154(2007) [119] --3------3------3----
BBa_S03154(2006) [120] ---3------3------3---
BBa_I9026(2007) [121] ----3------3------3--
BBa_I9026(2007) [122] -----3------3------3-
BBa_J04500(2007) [123] ------1------3------3
EcoRⅠ -------0.10.10.10.10.10.10.10.10.10.10.10.10.10.1
PstⅠ --------------0.10.10.10.10.10.10.1
Buffer EcoRⅠ -------0.90.90.90.90.90.90.90.80.80.80.80.80.80.8
BSA -------11111111111111
dH2O -------55555555555555
TOTAL 33333311010101010101010101010101010


--->Gel Check
Chiba-0823-1.JPG
Sample No 1~67
Sample DNA 31
Loading Dye 11
dH2O 24
TOTAL 66
From left; Sanple No. 1~7



Chiba-0823-2.JPG
Sample No 8~21
Sample DNA 10
Loading Dye 2
TOTAL 12
From left;
(Single Digestion) Sanple No. 8~14
  • BBa_S03154(2007) [131] -> OK
  • BBa_S03154(2006) [132] -> OK
  • BBa_I9026(2007) [133] -> OK
  • BBa_I9026(2006) [134] -> OK
  • BBa_I9030(2007) [135] -> OK
  • BBa_I9030(2006) [136] -> OK
  • BBa_J04500(2007) [137] -> OK
(Double Digestion) Sample No. 15~21
  • BBa_S03154(2007) [138] -> OK
  • BBa_S03154(2006) [139] -> OK
  • BBa_I9026(2007) [140] -> OK
  • BBa_I9026(2006) [141] -> OK
  • BBa_I9030(2007) [142] -> OK
  • BBa_I9030(2006) [143] -> OK
  • BBa_J04500(2007) [144] -> OK?



Digestion test
BBa_J04500(2007:old-sample)[145]
Sample No 123
Sample DNA 333
EcoRⅠ -0.10.1
SpeⅠ --0.1
EcoRⅠ Buffer -0.90.8
BSA -11
dH2O -55
TOTAL 31010


--->Gel Check

Chiba-0823-3.JPG
Sample No 123
Sample DNA 31010
Loading Dye 122
dH2O 2--
TOTAL 61212
From left;
  1. BBa_J04500(2007) [146] -> OK
  2. BBa_J04500(2007) [147]Single Digestion -> OK
  3. BBa_J04500(2007) [148]Double Digestion -> Bad

Week 2

24 August, 2008

(22/8)--->Mini prep
  1. BBa_J04500[149](2007)
  2. BBa_J04500[150](2006)
  3. BBa_J13002[151](2007)
  4. BBa_J13002[152](2006)


--->Digestion test
  1. BBa_J04500[153](2007)
  2. BBa_J04500[154](2006)
  3. BBa_J13002[155](2007)
  4. BBa_J13002[156](2006)


Sample No 5678
BBa_J04500[157](2007) 1---
BBa_J04500[158](2006) -1--
BBa_J13002[159](2007) --1-
BBa_J13002[160](2007) ---1
EcoRⅠ 0.10.10.10.1
Buffer 4 0.90.90.90.9
dH2O 8888
TOTAL 10101010


--->Gel Check
Chiba-0824-1.JPG
Sample No 1~8
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
  1. BBa_J04500[161](2007) -> ?
  2. BBa_J04500[162](2006) -> OK
  3. BBa_J13002[163](2007) -> OK
  4. BBa_J13002[164](2006) -> OK
  5. BBa_J04500[165](2007) -> OK
  6. BBa_J04500[166](2006) -> OK
  7. BBa_J13002[167](2007) -> OK
  8. BBa_J13002[168](2006) -> None?
Chiba-0824-2.JPG
Sample No 5~8
Sample DNA 9
Loading Dye 2
dH2O 1
TOTAL 12

From the left; (single digestuion)

5, BBa_J04500[169](2007) -> OK
6, BBa_J04500[170](2006) -> OK
7, BBa_J13002[171](2007) -> 3400bp:Bad, 2200bp:OK
8, BBa_J13002[172](2006) -> OK

25 August, 2008

(24/8)--->Digestion
  1. Double Digestion:BBa_J04500[173](2007)
  2. Double Digesiton:BBa_R0010[174](2007)
  3. Single Digestion:BBa_R0010[175](2007)
  4. BBa_R0010[176](2007)
Sample No 1234
Sample DNA 5511
EcoRⅠ 0.5o.5--
SpeⅠ 0.50.5--
Buffer 1 11--
Buffer 3 --1-
BSA 11--
dH2O 227.5-
TOTAL 1010101
--->Gel Check
Chiba-0825-1.JPG
Sample No 1~34
Sample DNA 101
Loading Dye 21
dH2O -4
TOTAL 126
From right;
  1. Double Digestion:BBa_J04500[177](2007) -> OK
  2. Double Digesiton:BBa_R0010[178](2007) -> ?(low)
  3. Single Digestion:BBa_R0010[179](2007) -> ?(low)
  4. BBa_R0010[180](2007) -> OK(low)


--->Digestion (Double Digestion)

  1. BBa_C0178[181]
  2. BBa_C0170[182]
  3. BBa_J04500[183](2007)
]

</tr>

Sample No 123
Sample DNA 33ng/μl×60μl33ng/μl×60μl100ng/μl×20μl
PstⅠ 222
XbaⅠ 22-
SpeⅠ --2
Buffer 2 --5
Buffer 3 1010-
BSA 10105
dH2O 161616
TOTAL 10010052

26 August, 2008

(8/25)--->Gel Check
Chiba-0825-2.JPG
Sample No 1~3
Sample DNA 30
Loading Dye 6
TOTAL 36
1, BBa_C0178[184]
Insert:609bp -> OK
Chiba-0825-3.JPG
2, BBa_C0170[185]
Insert:609bp -> OK
Chiba-0825-4.JPG
3, BBa_J04500[186](2007)
-> OK


--->Gel Extract


--->Zymo Clean
  1. 5μl
  2. 5μl
  3. 27μl


--->Gel Check
Chiba-0825-5.JPG
Sample No 1~3
Sample DNA 2
Loading Dye 2
H2O 8
TOTAL 12
From left;
  1. insert:C0178 -> OK
  2. insert:C0170 -> OK?
  3. vector:J04500 -> OK


--->SAP
Sample No 3
Sample DNA 26
SAP 1
SAP Buffer 3
TOTAL 30μL


--->Zymo Clean
Sample No.3->9μl


--->Gel Check
Chiba-0826.JPG
Sample No.3 1
Loading Dye 1
dH2O 4
TOTAL 6
Sample No.3 -> OK ・・・30ng?


--->Ligation
]

</tr>

Sample No (1)(2)(3)(4)(5)
insert①C0178 1--1-
insert②C0170 -1--1
vector③J04500 --222
ligase 11111
Buffer 22222
dH2O 66544
TOTAL 1010101010


--->Transformation
  1. insert①C0178
  2. insert②C0170
  3. vector③J04500
  4. insert①C0178+vector③J04500
  5. insert②C0170+vector③J04500


27 August, 2008

Digestion Test

Sample DNA : BBa_R0010[187]
Sample No 123
Sample DNA 111
PstⅠ --0.5
EcoRⅠ -0.50.5
Buffer 3 -11
BSA --1
dH2O -7.52
TOTAL 11010


--->Gel Check
Chiba-0827.JPG
Sample No 123
Sample DNA 155
Loading Dye 111
H2O 4--
TOTAL 666
From left;
  1. BBa_R0010
  2. Single Digestion
  3. Double Digestion


(26/8)--->Colony-PCR
Colony PCR of 5 colonies from ligation plates (26/8-(4),(5)) and one from control plate(BBa_F2620[188](2007)).
DNA Template 1
dNTP mix 5
Foward Primer 5
Reverse Primer 5
DNA polymerase 0.5
Thermopol Buffer 5
dH2O 28.5
TOTAL 50μL


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



--->Gel Check
Chiba-0827-2.JPG
Sample DNA 10
Loading Dye 2
TOTAL 12
From left;
1~5 insert:C0170 + vector:J04500 -> OK
6 F2620・・Positive Control -> OK
7~11 insert:C0178 + vector:J04500 -> OK


--->(28/8)Miniprep


Transformation

competent cells : XL10G


--->Mini prep


--->(29/8)Gel Check


28 August, 2008

(27/8)--->Mini prep
  1. insert:C0170 + vector:J04500
  2. insert:C0178 + vector:J04500


--->Digestion Test
  1. insert:C0170 + vector:J04500
  2. insert:C0178 + vector:J04500
Sample No 3456
insert:C0170 + vector:J04500 1-1-
insert:C0178 + vector:J04500 -1-1
EcoRⅠ 0.10.10.10.1
SpeⅠ --0.10.1
Buffer 3 1111
BSA --11
dH2O 7.87.86.86.8
TOTAL 10101010


--->(29/8)Gel Check
Chiba-0828.JPG
Sample No 1,23~6
Sample DNA 110
Loading Dye 12
H2O 4-
TOTAL 612
From left;
  1.  -> OK
  2.  -> OK
  3.  -> OK
  4.  -> OK
  5.  -> OK
  6.  -> OK

29 August, 2008

--->Digestion Test
  1. BBa_R0010(2007) [199]
  2. BBa_C0062(2007) [200]
  3. BBa_C0179(2007) [201]
  4. BBa_C0179(2006) [202]
  5. BBa_S03156(2007) [203]
  6. BBa_S03156(2006) [204]
  7. BBa_S03158(2007) [205]
  8. BBa_S03158(2006) [206]
  9. BBa_S03160(2007) [207]
  10. BBa_S03160(2006) [208]


Sample No S1~10W1W2~10
Sample DNA 142
PstⅠ -0.0750.1
EcoRⅠ 0.10.0750.1
Buffer 3 10.751
BSA -0.751
dH2O 7.94.355.8
TOTAL 101010


--->Gel Check
Chiba-0829.JPG
Sample No. 1~10S1~S10,W1~W10
Sample DNA 110
Loading Dye 12
TOTAL 612
From left;
1~5,S1,S5,W1~W5
Chiba-0829-2.JPG
From left;
6~10,S6~S10,W6~W10


(27/8)--->Gel Check
Chiba-0829-3.JPG
Sample DNA 0.5
Loading Dye 1
dH2O 4.5
TOTAL 6μL
R0010 -> OK


Digestion

  1. BBa_I9026(2007) [209]
  2. BBa_I9030(2007) [210]
  3. BBa_S03154(2007) [211]
  4. BBa_R0010(2006) [212]


Sample No 1~34
Sample DNA 100ng/μL×20200ng/μL×30
PstⅠ 22
XbaⅠ 2-
SpeⅠ -2
Buffer 2 -5
Buffer 3 5-
BSA 55
dH2O 166
TOTAL 5050
--->Gel Check
Chiba-0829-4.JPG
Sample No. 1~34
Sample DNA 103
Loading Dye 22
dH2O -7
TOTAL 1212
insert-I9026 : 685bp -> None
Chiba-0829-5.JPG
insert-I9030 : 745bp -> None
Chiba-0829-6.JPG
insert-S03154 : 685bp -> None
Chiba-0829-7.JPG
vector-R0010 : 2079bp -> OK


--->Gel Extract


--->Zymo Clean
->15μL
--->SAP
Sample No 4
Sample DNA 15
SAP 2
SAP Buffer 3
dH2O 10
TOTAL 30μL


--->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

Transformation
competent cells : XL10G
--->(31/8)Mini prep


(29/8)--->Mini prep
  1. insert:C0170 + vector:J04500
  2. insert:C0178 + vector:J04500


--->Digestion Test
  • insert:C0170 + vector:J04500 -> Sample No.1~4
  • insert:C0178 + vector:J04500 -> Sample No.5~8
  • BBa_T9002(2007)[221] -> 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
Sample No.Single : 10~18Double : 19~27
Sample DNA15
XbaⅠ0.10.1
SpeⅠ-0.1
Buffer 211
BSA11
dH2O6.92.8
TOTAL1010


--->Gel Check
Chiba-0830.JPG
Sample No 1~910~1819~27
Sample DNA 11010
Loading Dye 122
dH2O 4--
TOTAL 61212
From left;
Sanple No.1~13
-> OK
Chiba-0830-2.JPG
From left;
Sample No.14~17,24~16
14 -> OK
15,16 -> Bad
17 -> None
24~16 -> Bad
Chiba-0830-3.JPG
From left;
Sample No.18,27,19~23
18,27 -> Bad
19~22 -> OK
23 -> OK??

Week 3

31 August, 2008

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

Digestion

  1. BBa_I9026[222](2007)
  2. BBa_I9030[223](2006)
  3. BBa_S03154[224](2007)
  4. BBa_R0010[225](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[226]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[227]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[228]①、②
  • Double Digestion Plac+RBS+RhlI 2~5
  • Double Digestion BBa_T9002[229]①、②
  • BBa_T9002[230]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

2 September, 2008

(31/8)--->Gel Check
Chiba-0902.JPG
Sample No
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[231](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[232](2007):Positive control -> OK


--->(3/9)Mini prep



(1/9)--->Transformation
Competent cells : BW ΔFliC 40μL
  • Plac+RBS+LasI : Sample No.L1~L4
  • Plac+RBS+RhlI : Sample No.R1~R4
--->(2/9)OD

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


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

4 September, 2008

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

Gel Check
  1. BBa_C0161[233](2007)
  2. BBa_J04500[234](2007)
  3. BBa_R0010[235](2007)
Chiba-0913.JPG
Sample No 1~3
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;


--->[Team:Chiba/protocol/digestion|Digestion]]

  1. BBa_C0161[236](2007)
  2. BBa_J04500[237](2007)
  3. BBa_R0010[238](2007)


Sample No
Sample DNA 5511
EcoRⅠ 0.5o.5--
SpeⅠ 0.50.5--
Buffer 1 11--
Buffer 3 --1-
BSA 11--
dH2O 227.5-
TOTAL 1010101

Week 5

14 September, 2008

(13/9)--->Mini prep


Transformation
Competent cells : XL10G
  • BBa_T9002[239](2007)
  • Plac+RhlI+LVA
  • Plac+CinI+LVA
--->(15/9)

15 September, 2008

16 September, 2008

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