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		+	[[Paris/October 3|yesterday]] -- [[Paris/October 5|tomorrow]] <br>
	== Minipreps ==		== Minipreps ==
Line 5:		Line 6:
	== Transformation of W121 and FR781 with pKD46 ==		== Transformation of W121 and FR781 with pKD46 ==
		+
		+	Classic protocol. we used 1µL of Miniprep into 100µL of TSS chemically competent W121 and FR781 bacteria.
	== Houston we got a problem, the upstream construction for wanner lose the Chloramphenicol resistance ==		== Houston we got a problem, the upstream construction for wanner lose the Chloramphenicol resistance ==
Line 35:		Line 38:
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D79		| style="background: #ccffcc;" |D79
-	|	+	|pJ23100>>lox71-B0015(T)
	|L44.1		|L44.1
	|Xba1		|Xba1
	|Pst1		|Pst1
	|		|
-	|	+	|BI
	|		|
	|		|
Line 46:		Line 49:
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D80		| style="background: #ccffcc;" |D80
-	|	+	|pJ23100>>lox71-B0015(T)
	|L44.2		|L44.2
	|Xba1		|Xba1
	|Pst1		|Pst1
	|		|
-	|	+	|BI
	|		|
	|		|
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	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D81		| style="background: #ccffcc;" |D81
-	|	+	|pJ23107>>lox71-B0015(T)
	|L45.1		|L45.1
	|Xba1		|Xba1
	|Pst1		|Pst1
	|		|
-	|	+	|BI
	|		|
	|		|
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	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D82		| style="background: #ccffcc;" |D82
-	|	+	|pJ23107>>lox71-B0015(T)
	|L45.2		|L45.2
	|Xba1		|Xba1
	|Pst1		|Pst1
	|		|
-	|	+	|BI
	|		|
	|		|
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	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D83		| style="background: #ccffcc;" |D83
-	|	+	|pTet>>lox71-B0015(T)
	|L46.1		|L46.1
	|Xba1		|Xba1
	|Pst1		|Pst1
	|		|
-	|	+	|BI
	|		|
	|		|
Line 90:		Line 93:
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D84		| style="background: #ccffcc;" |D84
-	|	+	|araC/pBad>>lox71-B0015(T)
	|L47.1		|L47.1
	|Xba1		|Xba1
	|Pst1		|Pst1
	|		|
-	|	+	|BI
	|		|
	|		|
Line 101:		Line 104:
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D85		| style="background: #ccffcc;" |D85
-	|	+	|B0015(T)-lox66-RBSDapAcoli
	|L53.7		|L53.7
	|EcoRI		|EcoRI
	|SpeI		|SpeI
	|		|
-	|	+	|FI
	|		|
	|		|
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	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D86		| style="background: #ccffcc;" |D86
-	|	+	|attB-lox66-RBSDapAcoli
	|L54.3		|L54.3
	|EcoRI		|EcoRI
	|SpeI		|SpeI
	|		|
-	|	+	|FI
	|		|
	|		|
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	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D87		| style="background: #ccffcc;" |D87
-	|	+	|attB-lox66-RBSDapAcoli
	|L54.4		|L54.4
	|EcoRI		|EcoRI
	|SpeI		|SpeI
	|		|
-	|	+	|FI
	|		|
	|		|
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	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D88		| style="background: #ccffcc;" |D88
-	|	+	|B0015(T)-lox66-RBSdapAsubtilis
	|L56.3		|L56.3
	|EcoRI		|EcoRI
	|SpeI		|SpeI
	|		|
-	|	+	|FI
	|		|
	|		|
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	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D89		| style="background: #ccffcc;" |D89
-	|	+	|attB-lox66-RBSdapAsubtilis
	|L57.1		|L57.1
	|EcoRI		|EcoRI
	|SpeI		|SpeI
	|		|
-	|	+	|FI
	|		|
	|		|
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	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D90		| style="background: #ccffcc;" |D90
-	|	+	|attB-lox66-RBSdapAsubtilis
	|L57.3		|L57.3
	|EcoRI		|EcoRI
	|SpeI		|SpeI
	|		|
-	|	+	|FI
	|		|
	|		|
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	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D91		| style="background: #ccffcc;" |D91
-	|	+	|<bbpart>BBa_J61025</bbpart>
	|MP14.1		|MP14.1
	|EcoRI		|EcoRI
	|XbaI		|XbaI
	|		|
-	|	+	|FV - Medium constitutive promoter in J61002 (cl. 1) <partinfo>BBa_J61025 SpecifiedComponents</partinfo>
	|		|
	|		|
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	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D92		| style="background: #ccffcc;" |D92
-	|	+	|<bbpart>BBa_J61025</bbpart>
	|MP14.2		|MP14.2
	|EcoRI		|EcoRI
	|XbaI		|XbaI
	|		|
-	|	+	|FV - Medium constitutive promoter in J61002 (cl. 2) <partinfo>BBa_J61025 SpecifiedComponents</partinfo>
	|		|
	|		|
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	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D93		| style="background: #ccffcc;" |D93
-	|	+	|<bbpart>BBa_J61025</bbpart>
	|MP14.1		|MP14.1
	|Spe1		|Spe1
	|Pst1		|Pst1
	|		|
-	|	+	|BV - Medium constitutive promoter in J61002 (cl. 1) <partinfo>BBa_J61025 SpecifiedComponents</partinfo>
	|		|
	|		|
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	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |D94		| style="background: #ccffcc;" |D94
-	|	+	|<bbpart>BBa_J61025</bbpart>
	|MP14.2		|MP14.2
	|Spe1		|Spe1
	|Pst1		|Pst1
	|		|
-	|	+	|BV - Medium constitutive promoter in J61002 (cl. 2) <partinfo>BBa_J61025 SpecifiedComponents</partinfo>
	|		|
	|		|
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	|2		|2
	|		|
-	|	+	|0
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |C20		| style="background: #ccffcc;" |C20
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	|2		|2
	|		|
-	|	+	|0
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |C21		| style="background: #ccffcc;" |C21
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	|2		|2
	|		|
-	|	+	|0
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |L65		| style="background: #ccffcc;" |L65
-	|D56	+	|D56 - araC/pBad>>RBS-Cre BBinsert
	|10		|10
-	|D72	+	|D72 - pSB2K3 BBvector
	|2		|2
	|		|
-	|	+	|0
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |L66		| style="background: #ccffcc;" |L66
-	|D75	+	|D75 - AraC-pBAD-RBS-CRE FI
	|10		|10
-	|D73	+	|D73 - pSB2K3 ES open vector
	|2		|2
	|		|
-	|	+	|0
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |L67		| style="background: #ccffcc;" |L67
-	|D59	+	|D59 - pTet>>lox71-gfptripart BI
	|10		|10
-	|D64	+	|D64 - FRT-CmR-FRT BBa_J61025 BV
	|2		|2
	|		|
-	|	+	|0
	|}		|}
	== Culture of S28, strain having the [Frt-CmR-Frt] contruction ==		== Culture of S28, strain having the [Frt-CmR-Frt] contruction ==
-	In order to test the Chloramphenicol resistance of this strain.	+	In order to test the Chloramphenicol resistance of these strains.<br>
		+	- S28.1<br>
		+	- S28.2
	== Transformation of MP14.1 and MP14.2 in DH5α ==		== Transformation of MP14.1 and MP14.2 in DH5α ==
	In order to test the Chloramphenicol resistance given by MP14.		In order to test the Chloramphenicol resistance given by MP14.

---

Latest revision as of 15:08, 11 October 2007

yesterday -- tomorrow

Contents 1 Minipreps 2 Transformation of W121 and FR781 with pKD46 3 Houston we got a problem, the upstream construction for wanner lose the Chloramphenicol resistance 4 Digestion reactions 5 Ligations 6 Culture of S28, strain having the [Frt-CmR-Frt] contruction 7 Transformation of MP14.1 and MP14.2 in DH5α

Minipreps

• from S49 = pKD46
• pY-2P-IntC

Transformation of W121 and FR781 with pKD46

Classic protocol. we used 1µL of Miniprep into 100µL of TSS chemically competent W121 and FR781 bacteria.

Houston we got a problem, the upstream construction for wanner lose the Chloramphenicol resistance

[Frt-CmR-Frt-pTet>>lox71-gfpTripart] (L58)

- First, we got an abnormally short length for the PCR with the wanner primers (about 1,7kb instead of 2,1kb; see October 2)
- Then i did a PCR on the same plasmid (L58) with the primers O18&O19. We got the same pattern of length (1,7kb and another one at about 3,5kb)
- To investigate the possibility to have lost 400kb in the CmR gene leading to the loss of resistence to Cm for the strain, i put some clones of L58 on plates with either Ampicilline or Chloramphenicol (10µg/mL).

---

> the clones grew on Amp but not on Cm...

it confirms the hypothesis of a loss inside the CmR gene.

---

> of course the results for the sequencing will give us the last word. They should come on Friday in late afternoon. We called the sequencing facility.

Digestion reactions

Digestion Products
Number	Product Name	Matrix Name	Enzyme 1	Enzyme 2	Size	Description
D79	pJ23100>>lox71-B0015(T)	L44.1	Xba1	Pst1		BI
D80	pJ23100>>lox71-B0015(T)	L44.2	Xba1	Pst1		BI
D81	pJ23107>>lox71-B0015(T)	L45.1	Xba1	Pst1		BI
D82	pJ23107>>lox71-B0015(T)	L45.2	Xba1	Pst1		BI
D83	pTet>>lox71-B0015(T)	L46.1	Xba1	Pst1		BI
D84	araC/pBad>>lox71-B0015(T)	L47.1	Xba1	Pst1		BI
D85	B0015(T)-lox66-RBSDapAcoli	L53.7	EcoRI	SpeI		FI
D86	attB-lox66-RBSDapAcoli	L54.3	EcoRI	SpeI		FI
D87	attB-lox66-RBSDapAcoli	L54.4	EcoRI	SpeI		FI
D88	B0015(T)-lox66-RBSdapAsubtilis	L56.3	EcoRI	SpeI		FI
D89	attB-lox66-RBSdapAsubtilis	L57.1	EcoRI	SpeI		FI
D90	attB-lox66-RBSdapAsubtilis	L57.3	EcoRI	SpeI		FI
D91	BBa_J61025	MP14.1	EcoRI	XbaI		FV - Medium constitutive promoter in J61002 (cl. 1)
D92	BBa_J61025	MP14.2	EcoRI	XbaI		FV - Medium constitutive promoter in J61002 (cl. 2)
D93	BBa_J61025	MP14.1	Spe1	Pst1		BV - Medium constitutive promoter in J61002 (cl. 1)
D94	BBa_J61025	MP14.2	Spe1	Pst1		BV - Medium constitutive promoter in J61002 (cl. 2)

Ligations

Ligations
Number	Insert	Insert Volume (µL)	Vector	Vector Volume (µL)	Comments	Number of colonies
C19			D72	2		0
C20			D73	2		0
C21			D64	2		0
L65	D56 - araC/pBad>>RBS-Cre BBinsert	10	D72 - pSB2K3 BBvector	2		0
L66	D75 - AraC-pBAD-RBS-CRE FI	10	D73 - pSB2K3 ES open vector	2		0
L67	D59 - pTet>>lox71-gfptripart BI	10	D64 - FRT-CmR-FRT BBa_J61025 BV	2		0

Culture of S28, strain having the [Frt-CmR-Frt] contruction

In order to test the Chloramphenicol resistance of these strains.
- S28.1
- S28.2

Transformation of MP14.1 and MP14.2 in DH5α

In order to test the Chloramphenicol resistance given by MP14.