From 2013.igem.org

(Difference between revisions)

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
Primer	141-49 SaApL	141-49 SaApL
Templete	Escherichia coli MG1655 red (Cell suspention)	(Cell suspention)

Result.

1-9: Fragment 3 - ApCm

Check PCR.

Fragment	(control)	Fragment 3 - ApCm	Fragment 3 - ApCm
Primer	2 sets of the others	141-1 141-20	141-1 141-74
Templete	Escherichia 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.

Fragment	Fragment 3 - ApCm
Primer	mhpA-CmC6 SaApL
Templete	Fragment 3 - Cm Ap(SaApR-SaApL)

Result.

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

Check PCR.

Fragment	Fragment 3 - Ap (control)	Fragment 3 - ApCm
Primer	CmNR-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.

Fragment	Fragment 3 - Ap (control)	Fragment 3 - ApCm
Primer	CmNR-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.

Fragment	Fragment 3 - ApCm (control)	Fragment 3 - ApCm
Primer	141-49 141-1	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

Fragment	Fragment 1-2-3	Fragment 1-2 (control)
Primer	CmNR-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

>>Go to next week Back to last week<< >>Go to next week

@@ Line 215: / Line 215: @@
 -19: Fragment 3 - AmCm<Br>
 <Br>
+<Br>
 <b>September 27th</b><Br>
-<b>September 28th</b><Br>
+Assay<Br>
+<PCR CHECK preculture>
+       Pythagorean devices
+<Table>
+<Tr><Td>LB 2ml + arabinose -10%  0.1ml</Td><Td> + with the inoculating loop 1655 ins①~③ No.1</Td></Tr>
+<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>
+<Tr><Td>LB 2ml + arabinose -10%  0.1ml </Td><Td>+                     1655</Td></Tr>
+<Tr><Td> LB 2ml                        </Td><Td>+ with the inoculating loop 1655 ins①~③ No.1</Td></Tr>
+<Tr><Td>LB 2ml                        </Td><Td> + with the inoculating loop 1655 ins①~③ No.2</Td></Tr>
+<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>
+</Table>
+                    ⇒I culture it at 39 degrees for 2h.
+<Br>
+<Br>
+<PCR CHECK Pythagorean devices FRT>
+<Table>
+<Tr><Td>Premix(1)</Td>	<Td>Cell suspension</Td>	<Td>ins①-③</Td>	ara(+) No.1~3</Td></Tr>
+<Tr><Td></Td>	<Td></Td>	<Td>Ins①-③</Td>	ara(-) No.1~3</Td></Tr>
+<Tr><Td></Td>	<Td></Td>	<Td>1655</Td>	ara(+)</Td></Tr>
+<Tr><Td></Td>	<Td></Td>	<Td>1655</Td>	ara(-)</Td></Tr>
+<Tr><Td></Td>	<Td>GXL Buff</Td>	<Td>20</Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td>dNTP</Td>	<Td>8</Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td>GXL</Td>	<Td>2</Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td></Td>	<Td> (30) </Td>	<Td></Td></Tr>
+<Tr><Td>Premix(2) </Td>	<Td>Premix(1) </Td>	<Td>27</Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td>CmNR-not2</Td>	<Td>9</Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td>141-1</Td>	<Td>9</Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td></Td>	<Td> (45) </Td>	<Td></Td></Tr>
+<Tr><Td>mix</Td>	<Td>Cell suspension</Td>	<Td>5λ</Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td>Premix(2) </Td>	<Td>5λ</Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td></Td>	<Td><4P3-3></Td>	<Td></Td></Tr>
+</Table>
+<Br>
+<Br>
+<PCR CHECK Ptet inversion>
+<Table>
+<Tr><Td>Cell suspension</Td>	<Td>1655</Td>	<Td>ins①~③</Td>	<Td>ara(+)</Td>	No.1~3</Td></Tr>
+<Tr><Td></Td>	<Td>1655</Td>	<Td>ins①~③</Td>	<Td>ara(-)</Td>	No.1~3</Td></Tr>
+<Tr><Td>Premix①</Td>	<Td>GXL Buffer</Td>	<Td>30</Td>	<Td></Td>	<Td></Td></Tr>
+<Tr><Td></Td><	<Td>dNTP</Td>	<Td>12</Td>	<Td></Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td>GXL</Td>	<Td>3</Td>	<Td></Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td></Td>	<Td> (45) </Td>	<Td></Td>	<Td></Td></Tr>
+<Tr><Td>Premix②</Td>	<Td></Td>	<Ptet1></Td>	<Ptet2></Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td>Premix(1) </Td>	<Td>21</Td>	<Td>21</Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td>FimE-C2-EcoRI</Td>	<Td>7</Td>	<Td>7</Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td>TetP1</Td>	<Td>7</Td>	<Td></Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td>TetP2</Td>	<Td></Td>	<Td>7</Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td></Td>	<Td> (35) </Td>	<Td> (35) </Td>	<Td></Td></Tr>
+<Tr><Td>mix</Td>	<Td>Premix②</Td>	<Td>5</Td>	<Td></Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td>Cell suspension</Td>	<Td>5</Td>	<Td></Td>	<Td></Td></Tr>
+<Tr><Td></Td>	<Td><4P3-3></Td>	<Td></Td>	<Td></Td>	<Td></Td></Tr>
+</Table>
+<Br>
+<Br>
+<plating assay Pythagorean devices>
+<Table>
+<Tr><Td>LB + arabinose (0.2%)</Td><Td>→</Td><Td>plating (single colony isolation)</Td></Tr>
+ <Tr><Td>                   </Td><Td>→</Td><Td>1655 + ins①~③     No.1~3</Td></Tr>
+<Tr><Td>                   </Td><Td>→</Td><Td>1655               No.1~3</Td></Tr>
+<Tr><Td>  LB + IPTG (0.4g/ml) </Td><Td>→</Td><Td> plating (single colony isolation) </Td></Tr>
+ <Tr><Td>                     </Td><Td>→</Td><Td>1655 + ins①~③     No.1~3</Td></Tr>
+<Tr><Td>                      </Td><Td>→</Td><Td>1655               No.1~3</Td></Tr>
+<Tr><Td> LB                </Td><Td>→</Td><Td>plating (single colony isolation) </Td></Tr>
+<Tr><Td>                </Td><Td>→</Td><Td>1655 + ins①~③     No.1~3</Td></Tr>
+<Tr><Td>                  </Td><Td>→</Td><Td>1655               No.1~3</Td></Tr>
+</Table>

Team:TMU-Tokyo/Notebook experiment/4th

From 2013.igem.org

Latest revision as of 01:25, 28 September 2013

Experiment > 4th week