Team:Chiba/jk/γ/week 5

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(New page: 実験ログ == Week 5 == == 7,September,2008 == <br>Cloning <br>Digestion <br>vector:BBa_R0040(ptet)① 75ng/μl <br>insert:BBa_S03158(RBS+RhlR)② 33.6ng/μl <br>ins...)
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[[Team:Chiba/jk|実験ログ]]
[[Team:Chiba/jk|実験ログ]]
== Week 5 ==
== Week 5 ==
-
 
-
== 7,September,2008 ==
 
-
 
-
<br>Cloning
 
-
<br>Digestion
 
-
<br>vector:BBa_R0040(ptet)① 75ng/μl
 
-
<br>insert:BBa_S03158(RBS+RhlR)② 33.6ng/μl
 
-
<br>insert:BBa_S03156(RBS+lasR)③ 33.6ng/μl
 
-
<br>insert:BBa_S03160(RBS+cinR)④ 38.4ng/μl
 
-
<br>vector:BBa_R0010(pLac)⑤ 100ng/μl
 
-
<br>insert:BBa_J52008(RBS+rLuc)⑥ 33.6ng/μl
 
-
<table width="250" border="4" 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>
 
-
</tr>
 
-
<tr>
 
-
<td>DNA(digestion sample)</td>
 
-
<td>50</td><td>54</td><td>54</td><td>45</td><td>14</td><td>54</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SpeⅠ</td>
 
-
<td>1</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</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><td>0.5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>XbaⅠ</td>
 
-
<td>0</td><td>0.5</td><td>0.5</td><td>0.5</td><td>0</td><td>0.5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA(x10)</td>
 
-
<td>0.6</td><td>7</td><td>7</td><td>6</td><td>2</td><td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer2</td>
 
-
<td>6</td><td>-</td><td>-</td><td>-</td><td>2</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer3</td>
 
-
<td>-</td><td>7</td><td>7</td><td>6</td><td>-</td><td>7<td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>1.9</td><td>1</td><td>1</td><td>2</td><td>0.5</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>60</td><td>70</td><td>70</td><td>60</td><td>20</td><td>70</td>
 
-
</tr>
 
-
</table>
 
-
<br>----->37℃ 3hour
 
-
<br>----->Gel Extraction
 
-
<table width="250" border="4" 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>
 
-
</tr>
 
-
<tr>
 
-
<td>DNA(digestion sample)</td>
 
-
<td>6</td><td>10</td><td>10</td><td>10</td><td>4</td><td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td><td>2</td><td>2</td><td>2</td><td>1</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>4</td><td>-</td><td>-</td><td>-</td><td>1</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td><td>12</td><td>12</td><td>12</td><td>6</td><td>12</td>
 
-
</tr>
 
-
<tr>
 
-
<td>well</td>
 
-
<td>10</td><td>7</td><td>7</td><td>6</td><td>5</td><td>7</td>
 
-
</table>
 
-
<br>->DNA purification->①-④,⑥->7μl ⑤->6μl
 
-
 
-
 
-
== 8,Septmeber,2008 ==
 
-
 
-
<br>SAP
 
-
<br>BBa_R0040(ptet)①
 
-
<br>BBa_R0010(pLac)⑤
 
-
<table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①</td><td>⑤</td>
 
-
</tr>
 
-
<tr>
 
-
<td>DNA(digestion sample)</td>
 
-
<td>7</td><td>6</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SAP/td>
 
-
<td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SAP Buffer</td>
 
-
<td>3</td><td>3</td><td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>30</td><td>30</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>30</td><td>30</td>
 
-
</tr>
 
-
</table>
 
-
<br>->37℃ 1hour
 
-
<br>->65℃ 30min
 
-
<br>->DNA purification
 
-
<br>->21μl  6μl
 
-
<br>->Gel cheack
 
-
<table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>each</td>
 
-
</tr>
 
-
<tr>
 
-
<td>DNA(digestion sample)</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>
 
-
<br>vector:BBa_R0040(ptet)① 75ng/μl
 
-
<br>insert:BBa_S03158(RBS+RhlR)② 33.6ng/μl
 
-
<br>insert:BBa_S03156(RBS+lasR)③ 33.6ng/μl
 
-
<br>insert:BBa_S03160(RBS+cinR)④ 38.4ng/μl
 
-
<br>vector:BBa_R0010(pLac)⑤ 100ng/μl
 
-
<br>insert:BBa_J52008(RBS+rLuc)⑥ 33.6ng/μl
 
-
<table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①&②</td><td>①&③</td><td>①&④</td><td>①</td><td>⑤&⑥</td><td>⑤</td>
 
-
</tr>
 
-
<tr>
 
-
<td>DNA(vector)</td>
 
-
<td>3</td><td>3</td><td>3</td><td>3</td><td>5</td><td>2.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>DNA(insert)</td>
 
-
<td>3</td><td>3</td><td>3</td><td>-</td><td>3.1</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Ligase</td>
 
-
<td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Ligase Buffer(x5)</td>
 
-
<td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>1</td><td>1</td><td>1</td><td>4</td><td>8.9</td><td>4</td>
 
-
</tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td><td>10</td><td>10</td><td>20</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
== 9,September,2008 ==
 
-
 
-
 
-
 
-
== 10,September,2008 ==
 
-
 
-
<br>TIME Responce(liquid)
 
-
<br>TIME Responce(solid)
 
-
<br>Colony PCR
 
-
<br>->Gel cheack
 
-
<br>MINI Prep
 
-
 
-
 
-
== 13,September,2008 ==
 
-
 
-
<br>
 
Line 509: Line 315:
<br>ptet+RhlR(x5)
<br>ptet+RhlR(x5)
<br>ptet+cinR(x5)
<br>ptet+cinR(x5)
-
 

Revision as of 13:37, 25 September 2008

実験ログ

Contents

Week 5

14,September,2008


Digestion test
BBa_Q04510
BBa_I716463
BBa_C0051
BBa_C0070
BBa_J06620
BBa_J22141
BBa_C0076
BBa_I716542
BBa_I716462
BBa_C0078
BBa_J01101
BBa_C0071
BBa_I712019
BBa_S01416
BBa_C0079
BBa_C0077
BBa_J23007
BBa_I7100(x2)
BBa_S01416

Sample No each
DNA(digestion sample) 3
Buffer2(x10) 1
dH2O 6
EcoRⅠ 0.075
TOTAL 10


->37℃ 1hour
Gel cheack

Sample No each
DNA(digestion sample) 10
Loading Dye 2
TOTAL 12


16,September,2008


Digestion test
BBa_R0040(ptet)+BBa_S03158(RBS+RhlR)(x2)
BBa_R0040(ptet)+BBa_S03156(RBS+lasR)(x5)
BBa_R0040(ptet)+BBa_S03160(RBS+cinR)(x2)
BBa_S03158(RBS+RhlR)
BBa_S03156(RBS+lasR)
BBa_S03160(RBS+cinR)

</tr>

Sample No each
DNA 3
EcoRⅠ 0.083
PstⅠ 0.083
BSA(x10) 1
Buffer3(x10) 1
dH2O 5
TOTAL 10


->37℃ 1hour
Gel cheack

Sample No each
DNA 10
Loading Dye 2
TOTAL 12


TRANSFORATION
BBa_J37019(LuxI+RBS+LuxR)->no colony
BBa_J37003(RBS+LuxR)
BBa_J37020(LuxI+LuxR+pLux+GFP)
BBa_J37032(pLux+GFP)
PCR
Venus YFP(ptet+LuxR+YFP)

Sample No YFP
DNA 1
Venus Fwd 5
Rev (VR) 5
Thermo pol Buffer 10
dNTP 10
Vent 1
dH2O 68
TOTAL 100


17,September,2008


TRANSFORMATION
BBa_I13500(RBS+GFP)
BBa_J37019(Luxpr+LuxR)-->no colony
MINI Prep
BBa_J37033(RBS+LuxR)
BBa_J37020(Luxpr+LuxR+LuxR+GFP)
BBa_J37032(pLux+GFP)
Digestion
BBa_I7100(ptetR+RBS)
BBa_S03119(ptet+LuxR)
Ligation product(ptet+LasR)
Ligation product(ptet+RhlR)
Ligation product(ptet+cinR)

Sample No each
DNA template 25
EcoRⅠ 1
PstⅠ 1
BSA(x10) 4
EcoRⅠ Buffer 4
dH2O 5
TOTAL 40


-->37℃ 1hour
->Next day


18,September,2008


SAP 1μl
->37℃　1hour
->30min　65℃
Zymo ->25μl
Ligation
vector:BBa_I7100
insert:BBa_S03119(ptet+LuxR),Ligation product(ptet+LasR,ptet+RhlR,ptet+cinR)

Sample No Vector&insertVector
DNA(vector) 3.53.5
DNA(insert) 7.5-
Ligase 11
Ligase Buffer 44
dH2O 44
TOTAL 2020


TRANSFORMATION
BBa_J54103(RBS+GFP)
BBa_P2006


Gel Cheack
Ligation product(Venus YFP)
BBa_J37033
BBa_J37020
BBa_J37032

Sample No each
DNA each
Loading Dye 1
dH2O 4
TOTAL 6


19,September,2008


colony PCR
ptet+LuxR(x16)
ptet+LasR(x7)
ptet+RhlR(x5)
ptet+cinR(x5)



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