Team:TMU-Tokyo/Notebook experiment/4th

From 2013.igem.org

(Difference between revisions)
 
Line 218: Line 218:
<b>September 27th</b><Br>
<b>September 27th</b><Br>
Assay<Br>
Assay<Br>
 +
<PCR CHECK preculture>
       Pythagorean devices
       Pythagorean devices
-
<style>
+
<Table>
-
table {
+
<Tr><Td>LB 2ml + arabinose -10%  0.1ml</Td><Td> + with the inoculating loop 1655 ins①~③ No.1</Td></Tr>
-
border-collapse: collapse;
+
<Tr><Td>LB 2ml + arabinose -10%  0.1ml </Td><Td>+ with the inoculating loop 1655 ins①~③ No.2</Td></Tr>
-
}
+
<Tr><Td>LB 2ml + arabinose -10%  0.1ml</Td><Td> + with the inoculating loop 1655 ins①~③ No.3</Td></Tr>
-
th {
+
<Tr><Td>LB 2ml + arabinose -10%  0.1ml </Td><Td>+                     1655</Td></Tr>
-
border: solid 1px #666666;
+
<Tr><Td> LB 2ml                        </Td><Td>+ with the inoculating loop 1655 ins①~③ No.1</Td></Tr>
-
color: #000000;
+
<Tr><Td>LB 2ml                        </Td><Td> + with the inoculating loop 1655 ins①~③ No.2</Td></Tr>
-
background-color: #ff9999;
+
<Tr><Td>LB 2ml                        </Td><Td>+ with the inoculating loop 1655 ins①~③ No.3</Td></Tr>
-
}
+
<Tr><Td>LB 2ml                        </Td><Td> +                    1655</Td></Tr>
-
td {
+
</Table>
-
border: solid 1px #666666;
+
                    ⇒I culture it at 39 degrees for 2h.
-
color: #000000;
+
<Br>
-
background-color: #ffffff;
+
<Br>
-
}
+
<PCR CHECK Pythagorean devices FRT>
-
</style>
+
<Table>
-
<table>
+
 
-
<thead>
+
<Tr><Td>Premix(1)</Td> <Td>Cell suspension</Td> <Td>ins①-③</Td> ara(+) No.1~3</Td></Tr>
-
<tr>
+
<Tr><Td></Td> <Td></Td> <Td>Ins①-③</Td> ara(-) No.1~3</Td></Tr>
-
<th colspan="5">
+
<Tr><Td></Td> <Td></Td> <Td>1655</Td> ara(+)</Td></Tr>
-
<PCR CHECK preculture>
+
<Tr><Td></Td> <Td></Td> <Td>1655</Td> ara(-)</Td></Tr>
-
</th>
+
<Tr><Td></Td> <Td>GXL Buff</Td> <Td>20</Td> <Td></Td></Tr>
-
</tr>
+
<Tr><Td></Td> <Td>dNTP</Td> <Td>8</Td> <Td></Td></Tr>
-
</thead>
+
<Tr><Td></Td> <Td>GXL</Td> <Td>2</Td> <Td></Td></Tr>
-
<tbody>
+
<Tr><Td></Td> <Td></Td> <Td> (30) </Td> <Td></Td></Tr>
-
<tr>
+
<Tr><Td>Premix(2) </Td> <Td>Premix(1) </Td> <Td>27</Td> <Td></Td></Tr>
-
<th rowspan="2" colspan="9">
+
<Tr><Td></Td> <Td>CmNR-not2</Td> <Td>9</Td> <Td></Td></Tr>
-
      Pythagorean devices
+
<Tr><Td></Td> <Td>141-1</Td> <Td>9</Td> <Td></Td></Tr>
-
</th>
+
<Tr><Td></Td> <Td></Td> <Td> (45) </Td> <Td></Td></Tr>
-
</tr>
+
<Tr><Td>mix</Td> <Td>Cell suspension</Td> <Td>5λ</Td> <Td></Td></Tr>
-
<tr>
+
<Tr><Td></Td> <Td>Premix(2) </Td> <Td>5λ</Td> <Td></Td></Tr>
-
</tr>
+
<Tr><Td></Td> <Td></Td> <Td><4P3-3></Td> <Td></Td></Tr>
-
<tr>
+
</Table>
-
<th colspan="5">
+
<Br>
-
LB 2ml + arabinose -10%  0.1ml + with the inoculating loop 1655 ins①~③ No.1
+
<Br>
-
</th>
+
 
-
</tr>
+
 
-
<tr>
+
 
-
<th colspan="5">
+
 
-
                              +                                     No.2
+
 
-
</th>
+
 
-
</tr>
+
 
-
<tr>
+
<PCR CHECK Ptet inversion>
-
<th colspan="5">
+
<Table>
-
                              +                                     No.3
+
<Tr><Td>Cell suspension</Td> <Td>1655</Td> <Td>ins①~③</Td> <Td>ara(+)</Td> No.1~3</Td></Tr>
-
</th>
+
<Tr><Td></Td> <Td>1655</Td> <Td>ins①~③</Td> <Td>ara(-)</Td> No.1~3</Td></Tr>
-
</tr>
+
<Tr><Td>Premix①</Td> <Td>GXL Buffer</Td> <Td>30</Td> <Td></Td> <Td></Td></Tr>
-
<tr>
+
<Tr><Td></Td>< <Td>dNTP</Td> <Td>12</Td> <Td></Td> <Td></Td></Tr>
-
<th colspan="5">
+
<Tr><Td></Td> <Td>GXL</Td> <Td>3</Td> <Td></Td> <Td></Td></Tr>
-
1655
+
<Tr><Td></Td> <Td></Td> <Td> (45) </Td> <Td></Td> <Td></Td></Tr>
-
</th>
+
<Tr><Td>Premix②</Td> <Td></Td> <Ptet1></Td> <Ptet2></Td> <Td></Td></Tr>
-
</tr>
+
<Tr><Td></Td> <Td>Premix(1) </Td> <Td>21</Td> <Td>21</Td> <Td></Td></Tr>
-
<tr>
+
<Tr><Td></Td> <Td>FimE-C2-EcoRI</Td> <Td>7</Td> <Td>7</Td> <Td></Td></Tr>
-
<th colspan="5">
+
<Tr><Td></Td> <Td>TetP1</Td> <Td>7</Td> <Td></Td> <Td></Td></Tr>
-
LB 2ml                      + with the inoculating loop 1655 ins①~③ No.1
+
<Tr><Td></Td> <Td>TetP2</Td> <Td></Td> <Td>7</Td> <Td></Td></Tr>
-
</th>
+
<Tr><Td></Td> <Td></Td> <Td> (35) </Td> <Td> (35) </Td> <Td></Td></Tr>
-
</tr>
+
<Tr><Td>mix</Td> <Td>Premix②</Td> <Td>5</Td> <Td></Td> <Td></Td></Tr>
-
<tr>
+
<Tr><Td></Td> <Td>Cell suspension</Td> <Td>5</Td> <Td></Td> <Td></Td></Tr>
-
<th colspan="5">
+
<Tr><Td></Td> <Td><4P3-3></Td> <Td></Td> <Td></Td> <Td></Td></Tr>
-
                              +                                     No.2
+
</Table>
-
</th>
+
<Br>
-
</tr>
+
<Br>
-
<tr>
+
<plating assay Pythagorean devices>
-
<th colspan="5">
+
<Table>
-
                              +                                     No.3
+
<Tr><Td>LB + arabinose (0.2%)</Td><Td>→</Td><Td>plating (single colony isolation)</Td></Tr>
-
</th>
+
<Tr><Td>                   </Td><Td>→</Td><Td>1655 + ins①~③    No.1~3</Td></Tr>
-
</tr>
+
<Tr><Td>                   </Td><Td>→</Td><Td>1655               No.1~3</Td></Tr>
-
<tr>
+
<Tr><Td> LB + IPTG (0.4g/ml) </Td><Td>→</Td><Td> plating (single colony isolation) </Td></Tr>
-
<th colspan="5">
+
<Tr><Td>                     </Td><Td>→</Td><Td>1655 + ins①~③    No.1~3</Td></Tr>
-
1655
+
<Tr><Td>                     </Td><Td>→</Td><Td>1655               No.1~3</Td></Tr>
-
</th>
+
<Tr><Td> LB               </Td><Td>→</Td><Td>plating (single colony isolation) </Td></Tr>
-
</tr>
+
<Tr><Td>               </Td><Td>→</Td><Td>1655 + ins①~③    No.1~3</Td></Tr>
-
<tr>
+
<Tr><Td>                 </Td><Td>→</Td><Td>1655              No.1~3</Td></Tr>
-
<th rowspan="2" colspan="9">
+
</Table>
-
                    ⇒I culture it at 39 degrees for 2h.
+
 
-
</th>
+
 
-
</tr>
+
-
<tr>
+
-
</tr>
+
-
<tr>
+
-
<th rowspan="5" colspan="5">
+
-
<PCR CHECK Pythagorean devices FRT>
+
-
</th>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
Cell suspension
+
-
</td>
+
-
<td>
+
-
ins①-③
+
-
</td>
+
-
<td colspan="3">
+
-
ara(+) No.1~3
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
Ins①-③
+
-
</td>
+
-
<td colspan="3">
+
-
ara(-) No.1~3
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
1655
+
-
</td>
+
-
<td colspan="3">
+
-
ara(+)
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
1655
+
-
</td>
+
-
<td colspan="2">
+
-
ara(-)
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<th rowspan="4">
+
-
Premix(1)
+
-
</th>
+
-
<td>
+
-
GXL Buff
+
-
</td>
+
-
<td colspan="3">
+
-
20
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
dNTP
+
-
</td>
+
-
<td colspan="3">
+
-
8
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
GXL
+
-
</td>
+
-
<td colspan="4">
+
-
2
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td colspan="3">
+
-
-30
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<th rowspan="4">
+
-
Premix(2)
+
-
</th>
+
-
<td>
+
-
Premix(1)
+
-
</td>
+
-
<td colspan="3">
+
-
27
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
CmNR-not2
+
-
</td>
+
-
<td colspan="3">
+
-
9
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
141-1
+
-
</td>
+
-
<td colspan="4">
+
-
9
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td colspan="3">
+
-
-45
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<th rowspan="4">
+
-
mix
+
-
</th>
+
-
<td>
+
-
Cell suspension
+
-
</td>
+
-
<td colspan="3">
+
-
5λ
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
Premix(2)
+
-
</td>
+
-
<td colspan="4">
+
-
5λ
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td colspan="7">
+
-
<4P3-3>
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
</tr>
+
-
<tr>
+
-
<th colspan="5">
+
-
<PCR CHECK Ptet inversion>
+
-
</th>
+
-
</tr>
+
-
<tr>
+
-
<th rowspan="2">
+
-
Cell suspension
+
-
</th>
+
-
<td>
+
-
1655
+
-
</td>
+
-
<td>
+
-
ins①~③
+
-
</td>
+
-
<td>
+
-
ara(+)
+
-
</td>
+
-
<td>
+
-
No.1~3
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
1655
+
-
</td>
+
-
<td>
+
-
ins①~③
+
-
</td>
+
-
<td>
+
-
ara(-)
+
-
</td>
+
-
<td>
+
-
No.1~3
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<th rowspan="4">
+
-
Premix①
+
-
</th>
+
-
<td>
+
-
GXL Buffer
+
-
</td>
+
-
<td colspan="3">
+
-
30
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
dNTP
+
-
</td>
+
-
<td colspan="3">
+
-
12
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
GXL
+
-
</td>
+
-
<td colspan="4">
+
-
3
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td colspan="3">
+
-
-45
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<th rowspan="6" colspan="2">
+
-
Premix②
+
-
</th>
+
-
<td>
+
-
<Ptet1>
+
-
</td>
+
-
<td colspan="2">
+
-
<Ptet2>
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
Premix(1)
+
-
</td>
+
-
<td>
+
-
21
+
-
</td>
+
-
<td colspan="2">
+
-
21
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
FimE-C2-EcoRI
+
-
</td>
+
-
<td>
+
-
7
+
-
</td>
+
-
<td colspan="2">
+
-
7
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
TetP1
+
-
</td>
+
-
<td colspan="3">
+
-
7
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td colspan="2">
+
-
TetP2
+
-
</td>
+
-
<td colspan="3">
+
-
7
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
-35
+
-
</td>
+
-
<td colspan="2">
+
-
-35
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<th rowspan="4">
+
-
mix
+
-
</th>
+
-
<td>
+
-
Premix②
+
-
</td>
+
-
<td colspan="3">
+
-
5
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
Cell suspension
+
-
</td>
+
-
<td colspan="3">
+
-
5
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td colspan="8">
+
-
<4P3-3>
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
</tr>
+
-
<tr>
+
-
<th rowspan="2" colspan="9">
+
-
<plating assay Pythagorean devices>
+
-
</th>
+
-
</tr>
+
-
<tr>
+
-
</tr>
+
-
<tr>
+
-
<th colspan="5">
+
-
<plating assay Pythagorean devices>
+
-
</th>
+
-
</tr>
+
-
<tr>
+
-
<th colspan="5">
+
-
LB + arabinose (0.2%) → plating (single colony isolation)
+
-
</th>
+
-
</tr>
+
-
<tr>
+
-
<th colspan="5">
+
-
                          ・1655 + ins①~③    No.1~3
+
-
</th>
+
-
</tr>
+
-
<tr>
+
-
<th colspan="5">
+
-
                          ・1655               No.1~3
+
-
</th>
+
-
</tr>
+
-
<tr>
+
-
<th colspan="5">
+
-
LB + IPTG (0.4g/ml)   → plating (single colony isolation)
+
-
</th>
+
-
</tr>
+
-
<tr>
+
-
<th colspan="5">
+
-
                          ・1655 + ins①~③    No.1~3
+
-
</th>
+
-
</tr>
+
-
<tr>
+
-
<th colspan="5">
+
-
                          ・1655               No.1~3
+
-
</th>
+
-
</tr>
+
-
<tr>
+
-
<th colspan="5">
+
-
LB                   → plating (single colony isolation)
+
-
</th>
+
-
</tr>
+
-
<tr>
+
-
<th colspan="5">
+
-
                          ・1655 + ins①~③    No.1~3
+
-
</th>
+
-
</tr>
+
-
<tr>
+
-
<th rowspan="3" colspan="13">
+
-
                          ・1655              No.1~3
+
-
</th>
+
-
</tr>
+
-
<tr>
+
-
</tr>
+
-
<tr>
+
-
</tr>
+
-
</tbody>
+
-
</table>
+

Latest revision as of 01:25, 28 September 2013



Experiment > 4th week

September 22th
Mini prep following plasmid.
fimE-pBAD(in pSB1A3), pSB1C3-Km, pSB1A3-Km
Result.

1: pSB1C3-Km, 2-4: pSB1A3-Km, 5-6: fimE-pBAD


Check PCR.
Fragment(control)Fragment 3 - ApCm
Primer141-49
SaApL		141-49
SaApL
TempleteEscherichia coli
MG1655 red
(Cell suspention)		(Cell suspention)
Result.

1-9: Fragment 3 - ApCm

Check PCR.
Fragment(control)Fragment 3 - ApCmFragment 3 - ApCm
Primer2 sets of the others141-1
141-20		141-1
141-74
TempleteEscherichia coli
MG1655 red
(Cell suspention)		(Cell suspention)
Result.

1-7: 141-1 and 141-20, 8: control(141-1 and 141-20),
9-15: 141-1 and 141-74, 16: control(141-1 and 141-74),


Digestion following plasmid.
pSB1C3-Km-Blunt


P1 Preparation following fragment.
Fragment 1, 2


September 23th
PCR following fragment.
FragmentFragment 3 - ApCm
PrimermhpA-CmC6
SaApL
TempleteFragment 3 - Cm
Ap(SaApR-SaApL)
Result.

1: Fragment 3 - Cm, 2: Fragment 3 - ApCm,
3: pSB1C3-Km-Blunt


Check PCR.
FragmentFragment 3 - Ap
(control)		Fragment 3 - ApCm
PrimerCmNR-Not
SaApL		CmNR-Not
SaApL
Templete(Cell suspention)(Cell suspention)
Result.

1-31: Fragment 3 - ApCm


Electro Pration
Fragment 3 - Cm to Escherichia coli MG1655 red.


P1 Preparation
Fragment 3 - ApCm


September 24th
P1 transdunction
Fragment 1, 2
Result.



FragmentFragment 3 - Ap
(control)		Fragment 3 - ApCm
PrimerCmNR-Not
141-1		CmNR-Not
141-1
Templete(Cell suspention)(Cell suspention)
Result.

1-16: Fragment 3 - ApCm


P1 transduction
Fragment 2 to Fragment 1
Fragment 1 to Fragment 2
Result.


Get Fragment 1-2

Electro Poration
Fragment 1, 2, 3 - Ap, 3 - Cm, 3 - ApCm (in pSBC3-Km) to Escherichia coli DH1
Result.



September 25th
Check PCR.
FragmentFragment 3 - ApCm
(control)		Fragment 3 - ApCm
Primer141-49
141-1		CmNR-Not
141-1
Templete(Cell suspention)(Cell suspention)
Result.

1-7: 141-49 and 141-1, 8: control(141-49 and 141-8),
9-15: 141-1 and CmNR-Not, 16: control(141-1 and CmNR-Not)


September 26th
P1 transduction
Fragment 3 to Fragment 1-2
Get Fragment 1-2-3.


Check PCR
FragmentFragment 1-2-3Fragment 1-2
(control)
PrimerCmNR-Not
141-1		141-49
141-1
Templete(Cell suspention)(Cell suspention)
Result.

1-7: Fragment 1-2-3, 8: cotrol, 9-10: Fragment 1,
11-13: Fragment 2, 14-15: Fragment 3 - Ap, 16-17: Fragment 3 - Cm
18-19: Fragment 3 - AmCm


September 27th
Assay
Pythagorean devices
LB 2ml + arabinose -10% 0.1ml + with the inoculating loop 1655 ins①~③ No.1
LB 2ml + arabinose -10% 0.1ml + with the inoculating loop 1655 ins①~③ No.2
LB 2ml + arabinose -10% 0.1ml + with the inoculating loop 1655 ins①~③ No.3
LB 2ml + arabinose -10% 0.1ml + 1655
LB 2ml + with the inoculating loop 1655 ins①~③ No.1
LB 2ml + with the inoculating loop 1655 ins①~③ No.2
LB 2ml + with the inoculating loop 1655 ins①~③ No.3
LB 2ml + 1655
⇒I culture it at 39 degrees for 2h.

ara(+) No.1~3 ara(-) No.1~3 ara(+) ara(-)
Premix(1) Cell suspension ins①-③
Ins①-③
1655
1655
GXL Buff 20
dNTP 8
GXL 2
(30)
Premix(2) Premix(1) 27
CmNR-not2 9
141-1 9
(45)
mix Cell suspension 5λ
Premix(2) 5λ
<4P3-3>


No.1~3 No.1~3<
Cell suspension 1655 ins①~③ ara(+)
1655 ins①~③ ara(-)
Premix① GXL Buffer 30
dNTP 12
GXL 3
(45)
Premix②
Premix(1) 21 21
FimE-C2-EcoRI 7 7
TetP1 7
TetP2 7
(35) (35)
mix Premix② 5
Cell suspension 5
<4P3-3>


LB + arabinose (0.2%)→plating (single colony isolation)
→1655 + ins①~③ No.1~3
→1655 No.1~3
LB + IPTG (0.4g/ml) → plating (single colony isolation)
→1655 + ins①~③ No.1~3
→1655 No.1~3
LB →plating (single colony isolation)
→1655 + ins①~③ No.1~3
→1655 No.1~3


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