Team:Chiba/jk/β/week 3

From 2008.igem.org

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(→Week 3)
 
(46 intermediate revisions not shown)
Line 1: Line 1:
>[[Team:Chiba/jk/β|送受信班]]
>[[Team:Chiba/jk/β|送受信班]]
 +
 +
>[[Team:Chiba/protocol/j|Protocols]]
 +
==Week 3==
==Week 3==
Line 7: Line 10:
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
:Competent Cells : XL10G
:Competent Cells : XL10G
-
::*BBa_K084007(Plac+RBS+LasI)[http://partsregistry.org/Part:BBa_K084007]
+
::*[http://partsregistry.org/Part:BBa_K084007 BBa_K084007(Plac+RBS+LasI)]
-
::*BBa_K084008(Plac+RBS+RhlI)[http://partsregistry.org/Part:BBa_K084008]
+
::*[http://partsregistry.org/Part:BBa_K084008 BBa_K084008(Plac+RBS+RhlI)]
::---> We saved these plates with a refrigerator.
::---> We saved these plates with a refrigerator.
Line 14: Line 17:
:'''[[Team:Chiba/protocol/digestion|Digestion]]'''
:'''[[Team:Chiba/protocol/digestion|Digestion]]'''
-
::#BBa_I9026[http://partsregistry.org/Part:BBa_I9026](2007)
+
::#[http://partsregistry.org/Part:BBa_I9026 BBa_I9026](2007)
-
::#BBa_I9030[http://partsregistry.org/Part:BBa_I9030](2006)
+
::#[http://partsregistry.org/Part:BBa_I9030 BBa_I9030](2006)
-
::#BBa_S03154[http://partsregistry.org/Part:BBa_S03154](2007)
+
::#[http://partsregistry.org/Part:BBa_S03154 BBa_S03154](2007)
-
::#BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
+
::#[http://partsregistry.org/Part:BBa_R0010 BBa_R0010](2007)
:<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>1~3</td><td>4</td>
<td>1~3</td><td>4</td>
</tr>
</tr>
Line 50: Line 53:
</tr>
</tr>
<tr>
<tr>
-
<td>dH2O</td>
+
<td>dH<sub>2</sub>O</td>
<td>3.6</td><td>1.4</td></tr>
<td>3.6</td><td>1.4</td></tr>
<tr>
<tr>
Line 67: Line 70:
:--->'''[[Team:Chiba/protocol/digestion|Digestion test]]'''
:--->'''[[Team:Chiba/protocol/digestion|Digestion test]]'''
::*Plac+RBS+RhlI Sample No.2~5
::*Plac+RBS+RhlI Sample No.2~5
-
::*BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
+
::*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]①、②
<table width="600" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="600" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
Line 92: Line 95:
</tr>
</tr>
<tr>
<tr>
-
<td>dH2O</td>
+
<td>dH<sub>2</sub>O</td>
<td>7</td><td>5</td><td>5</td><td>7</td><td>5</td>
<td>7</td><td>5</td><td>5</td><td>7</td><td>5</td>
</tr>
</tr>
Line 109: Line 112:
:<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>Plac+RBS+RhlI Sample No.2~5, BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①②
+
<td>Plac+RBS+RhlI Sample No.2~5, [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]①②
</td><td>Single & Double Digestion</td>
</td><td>Single & Double Digestion</td>
</tr>
</tr>
Line 121: Line 124:
<td>1</td><td>2</td></tr>
<td>1</td><td>2</td></tr>
<tr>
<tr>
-
<td>dH2O</td>
+
<td>dH<sub>2</sub>O</td>
<td>4</td><td>-</td>
<td>4</td><td>-</td>
</tr>
</tr>
Line 131: Line 134:
:From Left
:From Left
::*Single Digestion Plac+RBS+RhlI 2~5
::*Single Digestion Plac+RBS+RhlI 2~5
-
::*Single Digestion BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
+
::*Single Digestion [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]①、②
::*Double Digestion Plac+RBS+RhlI 2~5
::*Double Digestion Plac+RBS+RhlI 2~5
-
::*Double Digestion BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
+
::*Double Digestion [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]①、②
-
::*BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
+
::*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]①、②
|}
|}
 +
 +
 +
===1 September, 2008===
===1 September, 2008===
Line 144: Line 150:
:<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>1~3</td><td>4</td>
<td>1~3</td><td>4</td>
</tr>
</tr>
Line 202: Line 208:
<td>1</td></tr>
<td>1</td></tr>
<tr>
<tr>
-
<td>dH2O</td>
+
<td>dH<sub>2</sub>O</td>
<td>4</td></tr>
<td>4</td></tr>
<tr>
<tr>
Line 212: Line 218:
::*insert-1(I9026) -> OK
::*insert-1(I9026) -> OK
::*insert-2(I9030) -> OK
::*insert-2(I9030) -> OK
-
::*insert-3(S03154) -> None --> Transformation BBa_S03154[http://partsregistry.org/Part:BBa_S03154]
+
::*insert-3(S03154) -> None --> Transformation [http://partsregistry.org/Part:BBa_S03154 BBa_S03154]-->
-
::*vector-4(R0010) -> OK
+
(2/9)'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''with [http://partsregistry.org/Part:BBa_K084009 BBa_K084009], [http://partsregistry.org/Part:BBa_K084010 BBa_K084010]
 +
::*vector-4(R0010)
|}
|}
Line 220: Line 227:
<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)</td><td>(2)</td><td>(3)</td><td>(4)</td><td>(5)</td>
<td>(1)</td><td>(2)</td><td>(3)</td><td>(4)</td><td>(5)</td>
</tr>
</tr>
Line 244: Line 251:
</tr>
</tr>
<tr>
<tr>
-
<td>dH2O</td>
+
<td>dH<sub>2</sub>O</td>
<td>2</td><td>2</td><td>5</td><td>5</td><td>5</td>
<td>2</td><td>2</td><td>5</td><td>5</td><td>5</td>
</tr>
</tr>
Line 257: Line 264:
:Competent cells : XL10GOLD  30μL
:Competent cells : XL10GOLD  30μL
:Transformed the following and grew on new ampicillin plates.  
:Transformed the following and grew on new ampicillin plates.  
-
::#BBa_K084009(Plac+RBS+RhlI+LVA, Amp)[http://partsregistry.org/Part:BBa_K084009] -> 628 colonies
+
::#[http://partsregistry.org/Part:BBa_K084009 BBa_K084009(Plac+RBS+RhlI+LVA, Amp)] -> 628 colonies
-
::#BBa_K084010(Plac+RBS+CinI+LVA, Amp)[http://partsregistry.org/Part:BBa_K084010] -> 500 colonies
+
::#[http://partsregistry.org/Part:BBa_K084010 BBa_K084010(Plac+RBS+CinI+LVA, Amp)] -> 500 colonies
::#insert-1(RBS+RhlI+LVA) -> 9 colonies
::#insert-1(RBS+RhlI+LVA) -> 9 colonies
::#insert-2(RBS+CinI+LVA) -> No colonies on the plate
::#insert-2(RBS+CinI+LVA) -> No colonies on the plate
Line 268: Line 275:
-
:--->'''[[Team:Chiba/protocol/transformation|Transformation]]'''
+
'''[[Team:Chiba/protocol/transformation|Transformation]]'''
-
:Competent cells : BW ΔFliC 40μL
+
:Competent cells : JW1908 40μL
:Transformed the following and grew on new ampicillin plates.  
:Transformed the following and grew on new ampicillin plates.  
-
::*BBa_K084007(Plac+RBS+LasI)[http://partsregistry.org/Part:BBa_K084007]
+
::*[http://partsregistry.org/Part:BBa_K084007 BBa_K084007(Plac+RBS+LasI)]
-
::*BBa_K084008(Plac+RBS+RhlI)[http://partsregistry.org/Part:BBa_K084008]
+
::*[http://partsregistry.org/Part:BBa_K084008 BBa_K084008(Plac+RBS+RhlI)]
-
::*BBa_T9002(Ptet+RBS+LuxR+GFP)[http://partsregistry.org/Part:BBa_K084010]
+
::*[http://partsregistry.org/Part:BBa_K084010 BBa_T9002(Ptet+RBS+LuxR+GFP)]
:--->(2/9)'''Liquid Culture'''
:--->(2/9)'''Liquid Culture'''
 +
 +
 +
===2 September, 2008===
===2 September, 2008===
Line 291: Line 301:
<td>1</td></tr>
<td>1</td></tr>
<tr>
<tr>
-
<td>dH2O</td>
+
<td>dH<sub>2</sub>O</td>
<td>4</td></tr>
<td>4</td></tr>
<tr>
<tr>
Line 307: Line 317:
:(1/9)--->'''[[Team:Chiba/protocol/PCR| Colony PCR]]'''
:(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)).
+
::Colony PCR of 8 colonies from ligation plates (1/9:(1)[http://partsregistry.org/Part:BBa_K084009 BBa_K084009](R1~R8),(2)[http://partsregistry.org/Part:BBa_K084010 BBa_K084010](C1~C8)) and one from control plate([http://partsregistry.org/Part:BBa_F2620 BBa_F2620](2007)).
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
Line 335: Line 345:
</tr>
</tr>
<tr>
<tr>
-
<td>dH2O</td>
+
<td>dH<sub>2</sub>O</td>
<td>20.5</td>
<td>20.5</td>
</tr>
</tr>
Line 349: Line 359:
--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
-
 
+
:{|align="justify"
-
{|align="justify"
+
|[[Image:Chiba-0902-2.JPG]]
|[[Image:Chiba-0902-2.JPG]]
|
|
Line 362: Line 371:
</tr>
</tr>
<tr>
<tr>
-
<td>dH2O</td>
+
<td>dH<sub>2</sub>O</td>
<td>4</td>
<td>4</td>
</tr>
</tr>
Line 394: Line 403:
:*Plac+RBS+CinI+LVA
:*Plac+RBS+CinI+LVA
::C7,C8 -> OK
::C7,C8 -> OK
-
:*BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007):Positive control -> OK
+
:*[http://partsregistry.org/Part:BBa_F2620 BBa_F2620](2007):Positive control -> OK
|}
|}
Line 403: Line 412:
-
:(1/9)--->'''Liquid Culture'''
+
(1/9)--->'''Liquid Culture'''
:Cultured the following cells (2mL LB-Amp, at 37℃, 7 hours)
:Cultured the following cells (2mL LB-Amp, at 37℃, 7 hours)
::from transformed plates:  
::from transformed plates:  
-
::*BBa_K084007(Plac+RBS+LasI, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_K084007]
+
::*[http://partsregistry.org/Part:BBa_K084007 BBa_K084007](Plac+RBS+LasI, Competent Cells : JW1908)
-
::*BBa_K084008(Plac+RBS+RhlI, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_K084008]
+
::*[http://partsregistry.org/Part:BBa_K084008 BBa_K084008](Plac+RBS+RhlI, Competent Cells : JW1908)
-
::*BBa_T9002(Ptet+RBS+LuxR+GFP, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_K084010]
+
::*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](Ptet+RBS+LuxR+GFP, Competent Cells : JW1908)
::from Glycerol Stock:
::from Glycerol Stock:
-
::*BBa_S03623(Ptet+RBS+LuxI, Competent Cells : BW ΔFliC)
+
::*[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](Ptet+RBS+LuxI, Competent Cells : JW1908)
-
:--->(3/9)'''Phenotype test'''
+
--->(3/9)'''Phenotype test'''
Line 418: Line 427:
'''[[Team:Chiba/protocol/transformation|Transformation]]'''
'''[[Team:Chiba/protocol/transformation|Transformation]]'''
:Competent cells : XL10G  30μL
:Competent cells : XL10G  30μL
-
::*C0161(2007) [http://partsregistry.org/Part:BBa_C0161]
+
::*[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007)
-
::*C0161(2006) [http://partsregistry.org/Part:BBa_C0161]
+
::*[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006)
-
::*C0261(2007) [http://partsregistry.org/Part:BBa_C0261]
+
::*[http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2007)
-
::*C0261(2006) [http://partsregistry.org/Part:BBa_C0261]
+
::*[http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2006)
-
:--->(4/9)'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
+
--->(4/9)'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
===3 September, 2008===
===3 September, 2008===
:(2/9)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
:(2/9)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 +
::*[http://partsregistry.org/Part:BBa_K084009 BBa_K084009](R1, R3~R7)
 +
::*[http://partsregistry.org/Part:BBa_K084010 BBa_K084010](C1,C2,C4~C8)
 +
::*[http://partsregistry.org/Part:BBa_S03154 BBa_S03154]
 +
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
Line 442: Line 455:
</tr>
</tr>
<tr>
<tr>
-
<td>dH2O</td>
+
<td>dH<sub>2</sub>O</td>
<td>4</td>
<td>4</td>
</tr>
</tr>
Line 451: Line 464:
</table>
</table>
:From left;
:From left;
-
::*S03154 -> OK
+
::*[http://partsregistry.org/Part:BBa_S03154 BBa_S03154] -> OK 33ng/μL
-
::*R1,R3~R7 (Plac+RBS+RhlI+LVA) -> OK
+
::*[http://partsregistry.org/Part:BBa_K084009 BBa_K084009](R1, R3~R7) -> OK
|}
|}
Line 459: Line 472:
|
|
:From left;
:From left;
-
::*C1,C2,C4~C8 (Plac+RBS+CinI+LVA) -> OK
+
::*[http://partsregistry.org/Part:BBa_K084010 BBa_K084010](C1,C2,C4~C8) -> OK
|}
|}
:--->'''[[Team:Chiba/protocol/digestion|Digestion test]]'''
:--->'''[[Team:Chiba/protocol/digestion|Digestion test]]'''
-
::*R1,R3~R7 (Plac+RBS+RhlI+LVA)
+
::*[http://partsregistry.org/Part:BBa_K084009 BBa_K084009](R1, R3~R7)
-
::*C1,C2,C4~C8 (Plac+RBS+CinI+LVA)
+
::*[http://partsregistry.org/Part:BBa_K084010 BBa_K084010](C1,C2,C4~C8)
<table width="500" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="500" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
Line 496: Line 509:
</tr>
</tr>
<tr>
<tr>
-
<td>dH2O</td>
+
<td>dH<sub>2</sub>O</td>
<td>7</td><td>5</td></tr>
<td>7</td><td>5</td></tr>
<tr>
<tr>
<td>TOTAL</td>
<td>TOTAL</td>
-
<td>10</td><td>10</td>
+
<td>10μL</td><td>10μL</td>
</tr>
</tr>
</table>
</table>
Line 523: Line 536:
</table>
</table>
:From left;
:From left;
-
:*Single Digestion : R1,R3~R7 -> OK
+
:*Single Digestion : [http://partsregistry.org/Part:BBa_K084009 BBa_K084009]R1,R3~R7 -> OK
-
:*Single Digestion : C1,C2,C4~C8 -> OK
+
:*Single Digestion : [http://partsregistry.org/Part:BBa_K084010 BBa_K084010]C1,C2,C4~C8 -> OK
|}
|}
Line 531: Line 544:
|
|
:From left;
:From left;
-
:*Double Digestion : R1,R3~R7 -> OK
+
:*Double Digestion : [http://partsregistry.org/Part:BBa_K084009 BBa_K084009]R1,R3~R7 -> OK
-
:*Double Digestion : C1,C2,C4~C8 -> OK
+
:*Double Digestion : [http://partsregistry.org/Part:BBa_K084010 BBa_K084010]C1,C2,C4~C8 -> OK
|}
|}
Line 538: Line 551:
:(2/9)--->'''Phenotype-test'''
:(2/9)--->'''Phenotype-test'''
:*MIX
:*MIX
 +
::*[http://partsregistry.org/Part:BBa_K084007 BBa_K084007](Plac+RBS+LasI, Competent Cells : JW1908) -> Sample Name : L1~L4
 +
::*[http://partsregistry.org/Part:BBa_K084008 BBa_K084008](Plac+RBS+RhlI, Competent Cells : JW1908) -> Sample Name : R1~R4
 +
::*[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](Ptet+RBS+LuxI, Competent Cells : JW1908)
 +
::*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](Ptet+RBS+LuxR+GFP, Competent Cells : JW1908)
 +
 +
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
<tr>
<tr>
Line 545: Line 564:
<tr>
<tr>
<td>L1</td>
<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>
+
<td>2mL</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td>
</tr>
</tr>
<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>L2</td><td>-</td><td>2mL</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>L3</td><td>-</td><td>-</td><td>2mL</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>L4</td><td>-</td><td>-</td><td>-</td><td>2mL</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>R1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1mL</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>R2</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1mL</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>R3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1mL</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>R4</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1mL</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_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>2mL</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(100μM)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1μL</td></tr>
-
<tr><td>AHL(100μM)</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>BBa_T9002[http://partsregistry.org/Part:BBa_T9002]</td><td>2mL</td><td>2mL</td><td>2mL</td><td>2mL</td><td>1mL</td><td>1mL</td><td>1mL</td><td>1mL</td><td>2mL</td><td>1mL</td><td>1mL</td></tr>
 +
<tr><td>IPTG(100μM)</td><td>1μL</td><td>1μL</td><td>1μL</td><td>1μL</td><td>1μL</td><td>1μL</td><td>1μL</td><td>1μL</td><td>-</td><td>-</td><td>-</td></tr>
</table>
</table>
-
:*Incubated for 8hours at 37 degrees  
+
*Incubated for 8hours at 37 degrees  
-
:*Spindown (max rpm, 3 min)
+
*Spindown (max rpm, 3 min)
-
:*The measurement of the intensity GFP by Masahiro Tominaga
+
*The measurement of the intensity GFP(visual judgment)
-
::<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</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>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>
<tr>
<tr>
<td>the intensity GFP</td>
<td>the intensity GFP</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>+</td><td>+</td><td>+</td><td>+</td><td>++</td><td>+</td><td>+++</td><td>-</td>
</tr>
</tr>
</table>
</table>
-
 
+
*Leaving for 12 at room temperture.
 +
*Resuspension
 +
*The measurement of the intensity of GFP by Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
 +
<table width="500" 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>the intensity GFP</td>
 +
<td>25.65</td><td>18.91</td><td>20.39</td><td>21.85</td><td>26.62</td><td>26.58</td><td>30.07</td><td>33.60</td><td>27.41</td><td>10.72</td><td>55.54</td>
 +
</tr>
 +
</table>
===4 September, 2008===
===4 September, 2008===
-
 
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
{|align="justify"
{|align="justify"
Line 585: Line 615:
:<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>dH<sub>2</sub>O</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>6μL</td>
</tr>
</tr>
</table>
</table>
:From left;
:From left;
-
:
+
::[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007) -> OK
-
:
+
::[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007) -> OK
 +
::[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006) -> OK
 +
::[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006) -> OK
|}
|}
{|align="justify"
{|align="justify"
|[[Image:Chiba-0904-2.JPG]]
|[[Image:Chiba-0904-2.JPG]]
|
|
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+
:From left;
 +
::[http://partsregistry.org/Part:BBa_C0161 BBa_C0261](2007) -> OK
 +
::[http://partsregistry.org/Part:BBa_C0161 BBa_C0261](2007) -> OK
 +
::[http://partsregistry.org/Part:BBa_C0161 BBa_C0261](2006) -> OK
 +
::[http://partsregistry.org/Part:BBa_C0161 BBa_C0261](2006) -> OK
 +
:
 +
|}
 +
 
 +
 
 +
:--->'''[[Team:Chiba/protocol/digestion|Digestion test]]'''
 +
 
 +
<table width="600" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
<tr>
<tr>
-
<td width="257">Sample No</td>
+
<td width="257">Sample</td>
-
<td>①</td><td>②</td><td>③</td><td>④</td>
+
<td>Single Digesiton</td><td>Double Digestion</td>
</tr>
</tr>
<tr>
<tr>
-
<td>Sample DNA</td>
+
<td>Sample DNA</td><td>1</td><td>2</td>
-
<td>3</td><td>3</td><td>3</td><td>3</td>
+
</tr>
</tr>
<tr>
<tr>
-
<td>Loading Dye</td>
+
<td>EcoRⅠ</td><td>0.05</td><td>0.05</td>
-
<td>2</td><td>2</td><td>2</td><td>2</td>
+
</tr>
 +
<tr>
 +
<td>PstⅠ</td><td>-</td><td>0.05</td>
 +
</tr>
 +
<tr>
 +
<td>Buffer 3</td>
 +
<td>1</td><td>1</td>
 +
</tr>
 +
<tr>
 +
<td>BSA</td>
 +
<td>1</td><td>1</td>
</tr>
</tr>
<tr>
<tr>
-
<td>dH2O</td>
+
<td>dH<sub>2</sub>O</td>
-
<td>7</td><td>7</td><td>7</td><td>7</td>
+
<td>6.95</td><td>5.9</td>
</tr>
</tr>
<tr>
<tr>
<td>TOTAL</td>
<td>TOTAL</td>
-
<td>12</td><td>12</td><td>12</td><td>12</td>
+
<td>10μL</td><td>10μL</td>
</tr>
</tr>
</table>
</table>
-
:From left;
 
-
:
 
-
:
 
-
|}
 
 +
 +
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
{|align="justify"
{|align="justify"
|[[Image:Chiba-0904-3.JPG]]
|[[Image:Chiba-0904-3.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>
 +
<td>TOTAL</td>
 +
<td>12μL</td></tr>
 +
</table>
|
|
 +
:From left;
 +
::Single Digestion
 +
::*[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007)
 +
::*[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007)
 +
::*[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006)
 +
::*[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006)
 +
::*[http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2007)
 +
::*[http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2007)
 +
::*[http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2006)
 +
::*[http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2006)
 +
::ladder
 +
::Double digestion
 +
::*[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007)
 +
::*[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007)
 +
::*[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006)
 +
::*[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006)
 +
::*[http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2007)
 +
::*[http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2007)
 +
::*[http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2006)
 +
::*[http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2006)
 +
|}
 +
 +
 +
 +
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 +
:Competent cells : JW1908
 +
:Transformed the following and grew on new ampicillin plates.
 +
::*[http://partsregistry.org/Part:BBa_K084009 BBa_K084009](R-3,4,6,7)
 +
::*[http://partsregistry.org/Part:BBa_K084010 BBa_K084010](C-1,2,6,7)
 +
::*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](2007)
 +
 +
:--->(5/9)'''Liquid Culture'''
 +
 +
===5 September, 2008===
 +
:(4/9)--->'''Liquid Culture'''
 +
:Cultured the following cells (2mL LB-Amp, at 37℃)
 +
::from transformed plates:
 +
::*[http://partsregistry.org/Part:BBa_K084009 BBa_K084009](Plac+RBS+RhlI+LVA, Competent Cells : JW1908)(r1, r3~r7)
 +
::*[http://partsregistry.org/Part:BBa_K084010 BBa_K084010](Plac+RBS+RhlI, Competent Cells : JW1908)(c1,c2,c4~c8)
 +
::from Glycerol Stock:
 +
::*[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](Ptet+RBS+LuxI, Competent Cells : JW1908)
 +
 +
:--->'''Phenotype test'''
 +
:*MIX
:<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</td><td>②</td><td>③</td><td>④</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>
<tr>
<tr>
-
<td>Sample DNA</td>
+
<td>r3</td>
-
<td>3</td><td>3</td><td>3</td><td>3</td>
+
<td>1mL</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>r4</td><td>-</td><td>1mL</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr>
 +
<tr><td>r6</td><td>-</td><td>-</td><td>1mL</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr>
 +
<tr><td>r7</td><td>-</td><td>-</td><td>-</td><td>1mL</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr>
 +
<tr><td>c1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1mL</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr>
 +
<tr><td>c2</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1mL</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr>
 +
<tr><td>c6</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1mL</td><td>-</td><td>-</td><td>-</td><td>-</td></tr>
 +
<tr><td>c7</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1mL</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>1mL</td><td>-</td><td>-</td></tr>
 +
<tr><td>AHL(100μM)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1μL</td></tr>
 +
<tr><td>BBa_T9002[http://partsregistry.org/Part:BBa_T9002]</td><td>1mL</td><td>1mL</td><td>1mL</td><td>1mL</td><td>1mL</td><td>1mL</td><td>1mL</td><td>1mL</td><td>1mL</td><td>1mL</td><td>1mL</td></tr>
 +
<tr><td>IPTG(100μM)</td><td>1μL</td><td>1μL</td><td>1μL</td><td>1μL</td><td>1μL</td><td>1μL</td><td>1μL</td><td>1μL</td><td>-</td><td>-</td><td>-</td></tr>
 +
</table>
 +
 
 +
 
 +
*Incubated for 8hours at 37 degrees
 +
*The measurement of the intensity GFP by Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
 +
<table width="500" 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>the intensity GFP (Frist run)</td>
 +
<td>15.21</td><td>16.44</td><td>16.34</td><td>16.63</td><td>9.720</td><td>9.703</td><td>9.685</td><td>9.874</td><td>19.95</td><td>16.59</td><td>71.04</td>
 +
</tr>
 +
<tr><td>the intensity GFP (Second run)</td>
 +
<td>17.85</td><td>19.19</td><td>19.28</td><td>19.14</td><td>9.981</td><td>10.16</td><td>10.14</td><td>10.41</td><td>23.94</td><td>17.61</td><td>79.53</td>
 +
</tr>
 +
</table>
 +
 
 +
 
 +
 +
*Spindown (max rpm, 3 min)
 +
*The measurement of the intensity GFP(visual judgment) (UV 312nm)
 +
<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>the intensity GFP</td>
 +
<td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>++</td><td>-</td>
 +
</tr>
 +
</table>
 +
 
 +
===6 September, 2008===
 +
'''[[Team:Chiba/protocol/PCR|Colony-PCR]]'''
 +
::PCR of BBa_I9030 and two control,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>1</td>
 +
</tr>
 +
<tr>
 +
<td>Thermopol Buffer</td>
 +
<td>5</td>
 +
</tr>
 +
<tr>
 +
<td>dH<sub>2</sub>O</td>
 +
<td>33</td>
 +
</tr>
 +
<tr>
 +
<td>TOTAL</td>
 +
<td>50μL</td>
 +
</tr>
 +
</table>
 +
 
 +
 
 +
:95℃,5min -> ( 95℃,1min -> 54℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃ store
 +
 
 +
 
 +
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 +
 
 +
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 +
<tr>
 +
<td width="257">Sample DNA</td>
 +
<td>1</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>dH<sub>2</sub>O</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>
 +
</table>
 +
:From left;<br>
 +
:1    I9030 without LVA  -> OK
 +
:2    I9030 Positive Control  -> OK
 +
:3    F2620 Positive control  -> OK
 +
 
 +
'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 +
Sample DNA : R0010
 +
:<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>dH<sub>2</sub>O</td>
 +
<td>4</td>
 +
</tr>
 +
<tr>
 +
<td>TOTAL</td>
 +
<td>6</td>
</tr>
</tr>
</table>
</table>
-
:From left;
 
-
:
 
-
:
 
-
|}
 
-
===5 September, 2008===
 
-
===6 September, 2008===
+
 
 +
 
 +
'''[[Team:Chiba/protocol/digestion|Digestion Test]]'''
 +
:Sample DNA:
 +
1:R0010,2:S03154,3:J04500,4:C0161,5:C0261
 +
 
 +
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 +
<td width="257">Sample No.</td>
 +
<td>1</td><td>2</td><td>3</td><td>4</td><td>5</td>
 +
<tr>
 +
<td>Sample DNA</td>
 +
<td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
 +
</tr>
 +
<tr>
 +
<td>PstⅠ</td>
 +
<td>0.5</td><td>0.5</td><td>0.5</td><td>0.5</td><td>0.5</td>
 +
</tr>
 +
<tr>
 +
<td>SpeⅠ</td>
 +
<td>0.5</td><td>-</td><td>1</td><td>-</td><td>-</td>
 +
</tr>
 +
<tr>
 +
<td>XbaⅠ</td>
 +
<td>-</td><td>0.5</td><td>-</td><td>0.5</td><td>0.5</td>
 +
</tr>
 +
<tr>
 +
<td>Buffer 2</td>
 +
<td>2</td><td>-</td><td>3</td><td>-</td><td>-</td>
 +
</tr>
 +
<tr>
 +
<td>Buffer 3</td>
 +
<td>-</td><td>6</td><td>-</td><td>6</td><td>6</td>
 +
</tr>
 +
<tr>
 +
<td>(10×)BSA</td>
 +
<td>2</td><td>0.6</td><td>3</td><td>-</td><td>-</td>
 +
</tr>
 +
<tr>
 +
<td>(100×)BSA</td>
 +
<td>-</td><td>-</td><td>-</td><td>0.6</td><td>0.6</td>
 +
</tr>
 +
<tr>
 +
<td>dH<sub>2</sub>O</td>
 +
<td>3</td><td>6.4</td><td>7</td><td>1.4</td><td>1.4</td>
 +
</tr>
 +
<tr>
 +
<td>TOTAL</td>
 +
<td>20</td><td>60</td><td>30</td><td>60</td><td>60</td>
 +
</tr>
 +
</table>
===[[Team:Chiba/jk/β/week 4|>next week]]===
===[[Team:Chiba/jk/β/week 4|>next week]]===
 +
 +
 +
{| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"
{| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"
!align="center"|[[Team:Chiba/j|ホーム]]
!align="center"|[[Team:Chiba/j|ホーム]]

Latest revision as of 04:05, 24 October 2008

>送受信班

>Protocols


Contents

Week 3

>last week

31 August, 2008

Transformation
Competent Cells : XL10G
  • [http://partsregistry.org/Part:BBa_K084007 BBa_K084007(Plac+RBS+LasI)]
  • [http://partsregistry.org/Part:BBa_K084008 BBa_K084008(Plac+RBS+RhlI)]
---> We saved these plates with a refrigerator.


Digestion
  1. [http://partsregistry.org/Part:BBa_I9026 BBa_I9026](2007)
  2. [http://partsregistry.org/Part:BBa_I9030 BBa_I9030](2006)
  3. [http://partsregistry.org/Part:BBa_S03154 BBa_S03154](2007)
  4. [http://partsregistry.org/Part:BBa_R0010 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
  • [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]①、②
Sample Single Digesiton 2~5Double Digestion 2~5Single Digestion T9002①Single Digesiton T9002②Double 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, [http://partsregistry.org/Part:BBa_T9002 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 [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]①、②
  • Double Digestion Plac+RBS+RhlI 2~5
  • Double Digestion [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]①、②
  • [http://partsregistry.org/Part:BBa_T9002 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)
--->Gel extract
--->zymo
insert-1(I9026) -> 7μL
insert-2(I9030) -> 7μL
insert-3(S03154) -> 7μL
vector-4(R0010) -> 15μL
--->SAP
vector-4(R0010)
--->Zymo
vector-4(R0010) -> 20μL


--->Gel Check
Chiba-0901-4.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
  • insert-1(I9026) -> OK
  • insert-2(I9030) -> OK
  • insert-3(S03154) -> None --> Transformation [http://partsregistry.org/Part:BBa_S03154 BBa_S03154]-->

(2/9)Mini prepwith [http://partsregistry.org/Part:BBa_K084009 BBa_K084009], [http://partsregistry.org/Part:BBa_K084010 BBa_K084010]

  • vector-4(R0010)


--->Ligation
Sample No. (1)(2)(3)(4)(5)
insert-1(I9026) 3-3--
insert-2(I9030) -3-3-
vector-4(R0010) 33--3
ligase 11111
Buffer 11111
dH2O 22555
TOTAL 1010101010


--->Transformation
Competent cells : XL10GOLD 30μL
Transformed the following and grew on new ampicillin plates.
  1. [http://partsregistry.org/Part:BBa_K084009 BBa_K084009(Plac+RBS+RhlI+LVA, Amp)] -> 628 colonies
  2. [http://partsregistry.org/Part:BBa_K084010 BBa_K084010(Plac+RBS+CinI+LVA, Amp)] -> 500 colonies
  3. insert-1(RBS+RhlI+LVA) -> 9 colonies
  4. insert-2(RBS+CinI+LVA) -> No colonies on the plate
  5. vector-4(Plac, Amp) -> 186 colonies


--->(2/9) Colony PCR


Transformation

Competent cells : JW1908 40μL
Transformed the following and grew on new ampicillin plates.
  • [http://partsregistry.org/Part:BBa_K084007 BBa_K084007(Plac+RBS+LasI)]
  • [http://partsregistry.org/Part:BBa_K084008 BBa_K084008(Plac+RBS+RhlI)]
  • [http://partsregistry.org/Part:BBa_K084010 BBa_T9002(Ptet+RBS+LuxR+GFP)]
--->(2/9)Liquid Culture



2 September, 2008

(31/8)--->Gel Check
Chiba-0902.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
I9026 -> OK, 100/μL
I9030 -> OK, 50ng/μL
S03154 -> OK, 30ng/μL (too low for the ligation:1/9 )


(1/9)---> Colony PCR
Colony PCR of 8 colonies from ligation plates (1/9:(1)[http://partsregistry.org/Part:BBa_K084009 BBa_K084009](R1~R8),(2)[http://partsregistry.org/Part:BBa_K084010 BBa_K084010](C1~C8)) and one from control plate([http://partsregistry.org/Part:BBa_F2620 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
  • [http://partsregistry.org/Part:BBa_F2620 BBa_F2620](2007):Positive control -> OK


--->(3/9)Mini prep



(1/9)--->Liquid Culture

Cultured the following cells (2mL LB-Amp, at 37℃, 7 hours)
from transformed plates:
  • [http://partsregistry.org/Part:BBa_K084007 BBa_K084007](Plac+RBS+LasI, Competent Cells : JW1908)
  • [http://partsregistry.org/Part:BBa_K084008 BBa_K084008](Plac+RBS+RhlI, Competent Cells : JW1908)
  • [http://partsregistry.org/Part:BBa_T9002 BBa_T9002](Ptet+RBS+LuxR+GFP, Competent Cells : JW1908)
from Glycerol Stock:
  • [http://partsregistry.org/Part:BBa_S03623 BBa_S03623](Ptet+RBS+LuxI, Competent Cells : JW1908)

--->(3/9)Phenotype test


Transformation

Competent cells : XL10G 30μL
  • [http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007)
  • [http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006)
  • [http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2007)
  • [http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2006)

--->(4/9)Mini prep

3 September, 2008

(2/9)--->Mini prep
  • [http://partsregistry.org/Part:BBa_K084009 BBa_K084009](R1, R3~R7)
  • [http://partsregistry.org/Part:BBa_K084010 BBa_K084010](C1,C2,C4~C8)
  • [http://partsregistry.org/Part:BBa_S03154 BBa_S03154]


--->Gel Check
Chiba-0903.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
  • [http://partsregistry.org/Part:BBa_S03154 BBa_S03154] -> OK 33ng/μL
  • [http://partsregistry.org/Part:BBa_K084009 BBa_K084009](R1, R3~R7) -> OK
Chiba-0903-2.JPG
From left;
  • [http://partsregistry.org/Part:BBa_K084010 BBa_K084010](C1,C2,C4~C8) -> OK
--->Digestion test
  • [http://partsregistry.org/Part:BBa_K084009 BBa_K084009](R1, R3~R7)
  • [http://partsregistry.org/Part:BBa_K084010 BBa_K084010](C1,C2,C4~C8)
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 10μL10μL


--->Gel Check
Chiba-0903-3.JPG
Sample DNA 10
Loading Dye 2
TOTAL 12
From left;
  • Single Digestion : [http://partsregistry.org/Part:BBa_K084009 BBa_K084009]R1,R3~R7 -> OK
  • Single Digestion : [http://partsregistry.org/Part:BBa_K084010 BBa_K084010]C1,C2,C4~C8 -> OK
Chiba-0903-4.JPG
From left;
  • Double Digestion : [http://partsregistry.org/Part:BBa_K084009 BBa_K084009]R1,R3~R7 -> OK
  • Double Digestion : [http://partsregistry.org/Part:BBa_K084010 BBa_K084010]C1,C2,C4~C8 -> OK


(2/9)--->Phenotype-test
  • MIX
  • [http://partsregistry.org/Part:BBa_K084007 BBa_K084007](Plac+RBS+LasI, Competent Cells : JW1908) -> Sample Name : L1~L4
  • [http://partsregistry.org/Part:BBa_K084008 BBa_K084008](Plac+RBS+RhlI, Competent Cells : JW1908) -> Sample Name : R1~R4
  • [http://partsregistry.org/Part:BBa_S03623 BBa_S03623](Ptet+RBS+LuxI, Competent Cells : JW1908)
  • [http://partsregistry.org/Part:BBa_T9002 BBa_T9002](Ptet+RBS+LuxR+GFP, Competent Cells : JW1908)


Sample No. 1234567891011
L1 2mL----------
L2-2mL---------
L3--2mL--------
L4---2mL-------
R1----1mL------
R2-----1mL-----
R3------1mL----
R4-------1mL---
BBa_S03154[http://partsregistry.org/Part:BBa_S03154]--------2mL--
AHL(100μM)----------1μL
BBa_T9002[http://partsregistry.org/Part:BBa_T9002]2mL2mL2mL2mL1mL1mL1mL1mL2mL1mL1mL
IPTG(100μM)1μL1μL1μL1μL1μL1μL1μL1μL---
  • Incubated for 8hours at 37 degrees
  • Spindown (max rpm, 3 min)
  • The measurement of the intensity GFP(visual judgment)
Sample No. 1234567891011
the intensity GFP +++++++++++++-


  • Leaving for 12 at room temperture.
  • Resuspension
  • The measurement of the intensity of GFP by Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
Sample No. 1234567891011
the intensity GFP 25.6518.9120.3921.8526.6226.5830.0733.6027.4110.7255.54

4 September, 2008

--->Gel Check
Chiba-0904.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6μL
From left;
[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007) -> OK
[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007) -> OK
[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006) -> OK
[http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006) -> OK
Chiba-0904-2.JPG
From left;
[http://partsregistry.org/Part:BBa_C0161 BBa_C0261](2007) -> OK
[http://partsregistry.org/Part:BBa_C0161 BBa_C0261](2007) -> OK
[http://partsregistry.org/Part:BBa_C0161 BBa_C0261](2006) -> OK
[http://partsregistry.org/Part:BBa_C0161 BBa_C0261](2006) -> OK


--->Digestion test
Sample Single DigesitonDouble Digestion
Sample DNA12
EcoRⅠ0.050.05
PstⅠ-0.05
Buffer 3 11
BSA 11
dH2O 6.955.9
TOTAL 10μL10μL


--->Gel Check
Chiba-0904-3.JPG


Sample DNA 10
Loading Dye 2
TOTAL 12μL
From left;
Single Digestion
  • [http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007)
  • [http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007)
  • [http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006)
  • [http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006)
  • [http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2007)
  • [http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2007)
  • [http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2006)
  • [http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2006)
ladder
Double digestion
  • [http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007)
  • [http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007)
  • [http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006)
  • [http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006)
  • [http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2007)
  • [http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2007)
  • [http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2006)
  • [http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2006)


Transformation
Competent cells : JW1908
Transformed the following and grew on new ampicillin plates.
  • [http://partsregistry.org/Part:BBa_K084009 BBa_K084009](R-3,4,6,7)
  • [http://partsregistry.org/Part:BBa_K084010 BBa_K084010](C-1,2,6,7)
  • [http://partsregistry.org/Part:BBa_T9002 BBa_T9002](2007)
--->(5/9)Liquid Culture

5 September, 2008

(4/9)--->Liquid Culture
Cultured the following cells (2mL LB-Amp, at 37℃)
from transformed plates:
  • [http://partsregistry.org/Part:BBa_K084009 BBa_K084009](Plac+RBS+RhlI+LVA, Competent Cells : JW1908)(r1, r3~r7)
  • [http://partsregistry.org/Part:BBa_K084010 BBa_K084010](Plac+RBS+RhlI, Competent Cells : JW1908)(c1,c2,c4~c8)
from Glycerol Stock:
  • [http://partsregistry.org/Part:BBa_S03623 BBa_S03623](Ptet+RBS+LuxI, Competent Cells : JW1908)
--->Phenotype test
  • MIX
Sample No. 1234567891011
r3 1mL----------
r4-1mL---------
r6--1mL--------
r7---1mL-------
c1----1mL------
c2-----1mL-----
c6------1mL----
c7-------1mL---
BBa_S03154[http://partsregistry.org/Part:BBa_S03154]--------1mL--
AHL(100μM)----------1μL
BBa_T9002[http://partsregistry.org/Part:BBa_T9002]1mL1mL1mL1mL1mL1mL1mL1mL1mL1mL1mL
IPTG(100μM)1μL1μL1μL1μL1μL1μL1μL1μL---


  • Incubated for 8hours at 37 degrees
  • The measurement of the intensity GFP by Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
Sample No. 1234567891011
the intensity GFP (Frist run) 15.2116.4416.3416.639.7209.7039.6859.87419.9516.5971.04
the intensity GFP (Second run) 17.8519.1919.2819.149.98110.1610.1410.4123.9417.6179.53


  • Spindown (max rpm, 3 min)
  • The measurement of the intensity GFP(visual judgment) (UV 312nm)
Sample No. 1234567891011
the intensity GFP ++++----+++-

6 September, 2008

Colony-PCR

PCR of BBa_I9030 and two control,BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007)).
DNA Template 1
dNTP mix 5
Foward Primer 5
Reverse Primer 5
DNA polymerase 1
Thermopol Buffer 5
dH2O 33
TOTAL 50μL


95℃,5min -> ( 95℃,1min -> 54℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃ store


--->Gel Check
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
1 I9030 without LVA -> OK
2 I9030 Positive Control -> OK
3 F2620 Positive control -> OK

Gel Check Sample DNA : R0010

Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6



Digestion Test

Sample DNA:

1:R0010,2:S03154,3:J04500,4:C0161,5:C0261

Sample No. 12345
Sample DNA 11111
PstⅠ 0.50.50.50.50.5
SpeⅠ 0.5-1--
XbaⅠ -0.5-0.50.5
Buffer 2 2-3--
Buffer 3 -6-66
(10×)BSA 20.63--
(100×)BSA ---0.60.6
dH2O 36.471.41.4
TOTAL 2060306060

>next week

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/week_3"