Team:Chiba/jk/γ/week 6
From 2008.igem.org
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- | [[Team:Chiba/jk|実験ログ]] | + | <br>>[[Team:Chiba/jk|実験ログ]] |
- | [[Team:Chiba/jk/γ|出力班]] | + | <br>>[[Team:Chiba/jk/γ|出力班]] |
+ | |||
+ | |||
== WEEK 6 == | == WEEK 6 == | ||
+ | |||
+ | |||
+ | == 21,September,2008 == | ||
== 22,September,2008 == | == 22,September,2008 == | ||
Line 14: | Line 19: | ||
<table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
<tr> | <tr> | ||
- | <td width="257" | + | <td width="257">DNA</td> |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<td>2</td> | <td>2</td> | ||
</tr> | </tr> | ||
Line 46: | Line 47: | ||
</tr> | </tr> | ||
</table> | </table> | ||
+ | |||
+ | <br>TRANSFORMATION | ||
+ | <br>BBa_I6107 | ||
+ | <br>BBa_I6206 | ||
+ | <br>BBa_I6101 | ||
+ | <br>BBa_I6042 | ||
+ | <br>BBa_I0462 | ||
+ | |||
<br>Digestion | <br>Digestion | ||
- | <br>Vector: | + | <br>Vector:BBa_R0062① |
- | <br>Insert:BBa_R0062 | + | <br>Insert:BBa_E0240② |
+ | <br>Vector:BBa_I7100③ | ||
+ | <br>Insert:BBa_R0062④ | ||
+ | <br>Insert:BBa_J37033⑤ | ||
+ | <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> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA</td> | ||
+ | <td>25</td><td>25</td><td>25</td><td>25</td><td>25</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoRⅠ</td> | ||
+ | <td>-</td><td>-</td><td>1</td><td>1</td><td>-</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SpeⅠ</td> | ||
+ | <td>1</td><td>-</td><td>1</td><td>1</td><td>-</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PstⅠ</td> | ||
+ | <td>1</td><td>1</td><td>1</td><td>-</td><td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>XbaⅠ</td> | ||
+ | <td>-</td><td>1</td><td>-</td><td>-</td><td>1</td> | ||
+ | </tr> | ||
+ | <td>BSA(x10)</td> | ||
+ | <td>4</td><td>4</td><td>4</td><td>-</td><td>4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoRⅠ Buffer(x10)</td> | ||
+ | <td>-</td><td>-</td><td>4</td><td>4</td><td>-</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer 2</td> | ||
+ | <td>4</td><td>-</td><td>-</td><td>-</td><td>-</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer 3</td> | ||
+ | <td>-</td><td>4</td><td>-</td><td>-</td><td>4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dH2O</td> | ||
+ | <td>5</td><td>5</td><td>5</td><td>9</td><td>5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TOTAL</td> | ||
+ | <td>40</td><td>40</td><td>40</td><td>40</td><td>40</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <br>->1hour 37℃ | ||
+ | <br>->SAP 1μl(Vector only) ->1hour 37℃ ->30min 65℃ | ||
+ | <br>->Zymo ->10μl | ||
+ | <br>Gel cheack | ||
+ | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
+ | <tr> | ||
+ | <td width="257">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> | ||
+ | |||
+ | <br>->③&④&⑤のLigationはしない | ||
+ | |||
+ | == 24,September,2008 == | ||
+ | |||
+ | <br>Ligation | ||
+ | <br>Vector:BBa_R0062① | ||
+ | <br>Insert:BBa_E0240② | ||
+ | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
+ | <tr> | ||
+ | <td width="257">Sample No</td> | ||
+ | <td>①&②</td><td>①</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA(vector)</td> | ||
+ | <td>1</td><td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA(insert)</td> | ||
+ | <td>3.2</td><td>-</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Ligase</td> | ||
+ | <td>1</td><td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Ligase Buffer</td> | ||
+ | <td>2</td><td>2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dH2O</td> | ||
+ | <td>2.5</td><td>6</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TOTAL</td> | ||
+ | <td>10</td><td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <br>->TRANSFORMATION | ||
+ | |||
+ | |||
+ | == 25,September,2008 == | ||
+ | <br>colony PCR | ||
+ | <br>Vector:BBa_R0062 Insert:BBa_E0240 | ||
+ | <br>Vector:BBa_R0062 | ||
+ | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
+ | <tr> | ||
+ | <td width="257">Sample No</td> | ||
+ | <td>each</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>culture</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Fwd primer</td> | ||
+ | <td>1.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Rev primer</td> | ||
+ | <td>1.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Taq(DNA pol)</td> | ||
+ | <td>0.2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dNTPmix</td> | ||
+ | <td>3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>thermo pol Buffer</td> | ||
+ | <td>3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dH2O</td> | ||
+ | <td>19.8</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TOTAL</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <br>->95℃ 5min ->(95℃ 1min 50℃ 30sec 72℃ 1min)x25 ->72℃ 10min | ||
+ | |||
+ | <br>Digestion Test | ||
+ | <br>BBa_I6206 | ||
+ | <br>BBa_I6101 | ||
+ | <br>BBa_I6042 | ||
+ | <br>BBa_I0462 | ||
+ | <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</td> | ||
+ | <td>2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoRⅠ</td> | ||
+ | <td>0.2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoRⅠ Buffer</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dH2O</td> | ||
+ | <td>7</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TOTAL</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <br>->30min 37℃ | ||
+ | <br>Gel cheack | ||
+ | <br>BBa_I6206(digestion product) | ||
+ | <br>BBa_I6101(digestion product) | ||
+ | <br>BBa_I6042(digestion product) | ||
+ | <br>BBa_I0462(digestion product) | ||
+ | <br>BBa_I0642(natural) | ||
+ | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
+ | <tr> | ||
+ | <td width="257">Sample No</td> | ||
+ | <td>each(digestion)</td><td>I0462(natural)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA</td> | ||
+ | <td>10</td><td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Loading Dye</td> | ||
+ | <td>2</td><td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dH2O</td> | ||
+ | <td>-</td><td>4</td> | ||
+ | <tr> | ||
+ | <td>TOTAL</td> | ||
+ | <td>12</td><td>6</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <br>Digestion | ||
+ | <br>BBa_I0462 | ||
+ | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
+ | <tr> | ||
+ | <td width="257">DNA</td> | ||
+ | <td>25</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>XbaⅠ</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PstⅠ</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer 3</td> | ||
+ | <td>4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BSA</td> | ||
+ | <td>4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dH2O</td> | ||
+ | <td>5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TOTAL</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <br>->3hour 37℃ | ||
+ | <br>->Zymo ->10μl | ||
+ | |||
+ | <br>Ligation | ||
<br>Vector:BBa_I7100 | <br>Vector:BBa_I7100 | ||
<br>Insert:BBa_R0062 | <br>Insert:BBa_R0062 | ||
- | <br>Insert: | + | <br>Insert:BBa_I0462 |
+ | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
+ | <tr> | ||
+ | <td width="257">I7100</td> | ||
+ | <td>1.3</td><td>1.3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>R0062</td> | ||
+ | <td>2</td><td>-</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>I0462</td> | ||
+ | <td>3.3</td><td>-</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Ligase</td> | ||
+ | <td>1</td><td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Ligase Buffer</td> | ||
+ | <td>2</td><td>2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dH2O | ||
+ | <td>0.4</td><td>5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TOTAL</td> | ||
+ | <td>10</td><td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <br>->3hour 37℃ | ||
+ | <br>->Transformation | ||
+ | |||
+ | == 26,September,2008 == | ||
+ | |||
+ | <br>TIME DELAY TEST | ||
+ | |||
+ | <br>colony PCR | ||
+ | <br>Ligation product(26,September)x7 | ||
+ | <br>Vector:BBa_R0062 | ||
+ | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
+ | <tr> | ||
+ | <td width="257">Sample No</td> | ||
+ | <td>each</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>culture</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Fwd primer</td> | ||
+ | <td>1.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Rev primer</td> | ||
+ | <td>1.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Taq(DNA pol)</td> | ||
+ | <td>0.2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dNTPmix</td> | ||
+ | <td>3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>thermo pol Buffer</td> | ||
+ | <td>3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dH2O</td> | ||
+ | <td>19.8</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TOTAL</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <br>->95℃ 5min ->(95℃ 1min 50℃ 30sec 72℃ 1min)x25 ->72℃ 10min | ||
+ | <br>->Gel cheack | ||
+ | <br>->MINI Prep | ||
+ | |||
+ | == 27,September,2008 == | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <br>>[[Team:Chiba/jk/γ/week 5|Last Week]] | ||
+ | <br>>[[Team:Chiba/jk/γ/week 7|Next Week]] | ||
Latest revision as of 15:48, 9 October 2008
Contents |
WEEK 6
21,September,2008
22,September,2008
Time delay
23,September,2008
Digestion test
BBa_E0240
DNA | 2 |
EcoRⅠ | 0.5 |
PstⅠ | 0.5 |
BSA(x10) | 1 |
EcoRⅠ Buffer(x10) | 1 |
dH2O | 5 |
TOTAL | 10 |
TRANSFORMATION
BBa_I6107
BBa_I6206
BBa_I6101
BBa_I6042
BBa_I0462
Digestion
Vector:BBa_R0062①
Insert:BBa_E0240②
Vector:BBa_I7100③
Insert:BBa_R0062④
Insert:BBa_J37033⑤
Sample No | ① | ② | ③ | ④ | ⑤ |
DNA | 25 | 25 | 25 | 25 | 25 |
EcoRⅠ | - | - | 1 | 1 | - |
SpeⅠ | 1 | - | 1 | 1 | - |
PstⅠ | 1 | 1 | 1 | - | 1 |
XbaⅠ | - | 1 | - | - | 1 | BSA(x10) | 4 | 4 | 4 | - | 4 |
EcoRⅠ Buffer(x10) | - | - | 4 | 4 | - |
Buffer 2 | 4 | - | - | - | - |
Buffer 3 | - | 4 | - | - | 4 |
dH2O | 5 | 5 | 5 | 9 | 5 |
TOTAL | 40 | 40 | 40 | 40 | 40 |
->1hour 37℃
->SAP 1μl(Vector only) ->1hour 37℃ ->30min 65℃
->Zymo ->10μl
Gel cheack
DNA | 1 |
Loading Dye | 1 |
dH2O | 4 |
TOTAL | 6 |
->③&④&⑤のLigationはしない
24,September,2008
Ligation
Vector:BBa_R0062①
Insert:BBa_E0240②
Sample No | ①&② | ① |
DNA(vector) | 1 | 1 |
DNA(insert) | 3.2 | - |
Ligase | 1 | 1 |
Ligase Buffer | 2 | 2 |
dH2O | 2.5 | 6 |
TOTAL | 10 | 10 |
->TRANSFORMATION
25,September,2008
colony PCR
Vector:BBa_R0062 Insert:BBa_E0240
Vector:BBa_R0062
Sample No | each |
culture | 1 |
Fwd primer | 1.5 |
Rev primer | 1.5 |
Taq(DNA pol) | 0.2 |
dNTPmix | 3 |
thermo pol Buffer | 3 |
dH2O | 19.8 |
TOTAL | 30 |
->95℃ 5min ->(95℃ 1min 50℃ 30sec 72℃ 1min)x25 ->72℃ 10min
Digestion Test
BBa_I6206
BBa_I6101
BBa_I6042
BBa_I0462
Sample No | each |
DNA | 2 |
EcoRⅠ | 0.2 |
EcoRⅠ Buffer | 1 |
dH2O | 7 |
TOTAL | 30 |
->30min 37℃
Gel cheack
BBa_I6206(digestion product)
BBa_I6101(digestion product)
BBa_I6042(digestion product)
BBa_I0462(digestion product)
BBa_I0642(natural)
Sample No | each(digestion) | I0462(natural) |
DNA | 10 | 1 |
Loading Dye | 2 | 1 |
dH2O | - | 4 |
TOTAL | 12 | 6 |
Digestion
BBa_I0462
DNA | 25 |
XbaⅠ | 1 |
PstⅠ | 1 |
Buffer 3 | 4 |
BSA | 4 |
dH2O | 5 |
TOTAL | 40 |
->3hour 37℃
->Zymo ->10μl
Ligation
Vector:BBa_I7100
Insert:BBa_R0062
Insert:BBa_I0462
I7100 | 1.3 | 1.3 |
R0062 | 2 | - |
I0462 | 3.3 | - |
Ligase | 1 | 1 |
Ligase Buffer | 2 | 2 |
dH2O | 0.4 | 5 |
TOTAL | 10 | 10 |
->3hour 37℃
->Transformation
26,September,2008
TIME DELAY TEST
colony PCR
Ligation product(26,September)x7
Vector:BBa_R0062
Sample No | each |
culture | 1 |
Fwd primer | 1.5 |
Rev primer | 1.5 |
Taq(DNA pol) | 0.2 |
dNTPmix | 3 |
thermo pol Buffer | 3 |
dH2O | 19.8 |
TOTAL | 30 |
->95℃ 5min ->(95℃ 1min 50℃ 30sec 72℃ 1min)x25 ->72℃ 10min
->Gel cheack
->MINI Prep
27,September,2008
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