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Revision as of 16:20, 25 July 2007 (view source) Eismoustique (Talk | contribs) (→Ligation & transformation reactions) ← Older edit		Latest revision as of 17:50, 7 October 2007 (view source) Nicolas C. (Talk | contribs)
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		+	[[Paris/July 24|yesterday]] -- [[Paris/July 26|tomorrow]] <br>
	==Digestions==		==Digestions==
Line 85:		Line 86:
	|width=35%| Comments		|width=35%| Comments
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
-	| style="background: #ccffcc;" |L1	+	| style="background: #ccffcc;" |L1'
	|D23.1 (AraC/pBad promoter FI)		|D23.1 (AraC/pBad promoter FI)
	|8.5		|8.5
	|D9.1 (J61002 ready for insertion of a FI)		|D9.1 (J61002 ready for insertion of a FI)
	|1.5		|1.5
-	|	+	|construct with pBad promotor regulating expression of mRFP (in order to test activity of pBad)
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
-	| style="background: #ccffcc;" |L2	+	| style="background: #ccffcc;" |L2'
	|D18 (BB dig. lox66DapAE.coli)		|D18 (BB dig. lox66DapAE.coli)
	|7		|7
	|D22.1 (pSB1A2 Eco, Pst)		|D22.1 (pSB1A2 Eco, Pst)
	|3		|3
-	|	+	|Insertion of lox66DapAE.coli construct as a biobrick in pSB1A2 plasmid
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
-	| style="background: #ccffcc;" |L3	+	| style="background: #ccffcc;" |L3'
	|D19 (BB dig. lox66DapAsubtilis)		|D19 (BB dig. lox66DapAsubtilis)
	|7		|7
	|D22.1 (pSB1A2 Eco, Pst)		|D22.1 (pSB1A2 Eco, Pst)
	|3		|3
-	|	+	|Insertion of lox66DapAsubtilis construct as a biobrick in pSB1A2 plasmid
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
-	| style="background: #ccffcc;" |L4	+	| style="background: #ccffcc;" |L4'
	|D20 (Lox66-DapAE.coli BI)		|D20 (Lox66-DapAE.coli BI)
	|8		|8
	|D1 (pJ23100 BV)		|D1 (pJ23100 BV)
	|2		|2
-	|	+	|lo66-DapAE.coli under regulation of strong promotor pJ23100
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
-	| style="background: #ccffcc;" |L5	+	| style="background: #ccffcc;" |L5'
	|D21 (lox66-DapAsubtilis BI)		|D21 (lox66-DapAsubtilis BI)
	|6.5		|6.5
	|D1 (pJ23100 BV)		|D1 (pJ23100 BV)
	|2 (+1.5µL H2O)		|2 (+1.5µL H2O)
-	|	+	|lox66-DapAsubtilis under regulation of strong promotor pJ23100
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
-	| style="background: #ccffcc;" |L6	+	| style="background: #ccffcc;" |L6'
	|D14.1 (Cre ORF)		|D14.1 (Cre ORF)
	|8		|8
	|D15.1 (B0030 BV)		|D15.1 (B0030 BV)
	|2		|2
-	|	+	|construction of the biobrick strong RBS(B0030)-Cre ORF
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
-	| style="background: #ccffcc;" |L7	+	| style="background: #ccffcc;" |L7'
	|D27 (Lox71-ftsZ BI)		|D27 (Lox71-ftsZ BI)
	|6		|6
	|D1 (pJ23100 BV)		|D1 (pJ23100 BV)
	|2 (+2µL H2O)		|2 (+2µL H2O)
-	|	+	|lox71-ftsZ under regulation of strong promotor pJ23100
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
-	| style="background: #ccffcc;" |L8	+	| style="background: #ccffcc;" |L8'
	|D27 (Lox71-ftsZ BI)		|D27 (Lox71-ftsZ BI)
	|6		|6
	|D3 (pJ23107 BV)		|D3 (pJ23107 BV)
	|2 (+2 H2O)		|2 (+2 H2O)
-	|	+	|lox71-ftsZ under regulation of promotor pJ23107 (intermediate strength)
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
-	| style="background: #ccffcc;" |L9	+	| style="background: #ccffcc;" |L9'
	|lox66 [O14+O15] (0.2µM)		|lox66 [O14+O15] (0.2µM)
	|2		|2
	|D22.1 (pSB1A2 Eco, Pst)		|D22.1 (pSB1A2 Eco, Pst)
	|2 (+6 H2O)		|2 (+6 H2O)
-	|	+	|lox66 cloned as a biobrick in pSB1A2
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
-	| style="background: #ccffcc;" |L10	+	| style="background: #ccffcc;" |L10'
	|lox71 [O16+O17] (0.2µM)		|lox71 [O16+O17] (0.2µM)
	|2		|2
	|D22.1 (pSB1A2 Eco, Pst)		|D22.1 (pSB1A2 Eco, Pst)
	|2 (+6 H2O)		|2 (+6 H2O)
-	|	+	|lox71 cloned as a biobrick
	|}		|}

---

Latest revision as of 17:50, 7 October 2007

yesterday -- tomorrow

Digestions

Digestion of purifications of PCR products

Digestion Products
Number	Product Name	Matrix Name	Enzyme 1	Enzyme 2	size	Description
D28	RBS-DapAColi BB	P8 (RBS-DapAColi)	EcoRI	PstI	~950	Biobrick
D29	RBS-DapAColi	P8 (RBS-DapAColi)	XbaI	PstI	~950	Backward insert
D30	RBS-DapASubtilis BB	P9 (RBS-DapASubtilis)	EcoRI	PstI	~950	Biobrick
D31	RBS-DapASubtilis	P9 (RBS-DapASubtilis)	XbaI	PstI	~950	Backward insert
D32	Lox71-FtsA-FtsZ	P10 Lox71-FtsA-FtsZ	XbaI	PstI	~2600	Backward insert - EcoRI site still presents : we can't use EcoRI

Ligation & transformation reactions

The Ligation reactions performed yesterday have all failed (the reactions were performed at 42° by mistake, then, another 1µL of ligase was added for a 5min rxn at 25°)

The following reactions were performed today

Ligations
Number	Insert	Insert Volume (µL)	Vector	Vector Volume (µL)	Comments
L1'	D23.1 (AraC/pBad promoter FI)	8.5	D9.1 (J61002 ready for insertion of a FI)	1.5	construct with pBad promotor regulating expression of mRFP (in order to test activity of pBad)
L2'	D18 (BB dig. lox66DapAE.coli)	7	D22.1 (pSB1A2 Eco, Pst)	3	Insertion of lox66DapAE.coli construct as a biobrick in pSB1A2 plasmid
L3'	D19 (BB dig. lox66DapAsubtilis)	7	D22.1 (pSB1A2 Eco, Pst)	3	Insertion of lox66DapAsubtilis construct as a biobrick in pSB1A2 plasmid
L4'	D20 (Lox66-DapAE.coli BI)	8	D1 (pJ23100 BV)	2	lo66-DapAE.coli under regulation of strong promotor pJ23100
L5'	D21 (lox66-DapAsubtilis BI)	6.5	D1 (pJ23100 BV)	2 (+1.5µL H2O)	lox66-DapAsubtilis under regulation of strong promotor pJ23100
L6'	D14.1 (Cre ORF)	8	D15.1 (B0030 BV)	2	construction of the biobrick strong RBS(B0030)-Cre ORF
L7'	D27 (Lox71-ftsZ BI)	6	D1 (pJ23100 BV)	2 (+2µL H2O)	lox71-ftsZ under regulation of strong promotor pJ23100
L8'	D27 (Lox71-ftsZ BI)	6	D3 (pJ23107 BV)	2 (+2 H2O)	lox71-ftsZ under regulation of promotor pJ23107 (intermediate strength)
L9'	lox66 [O14+O15] (0.2µM)	2	D22.1 (pSB1A2 Eco, Pst)	2 (+6 H2O)	lox66 cloned as a biobrick in pSB1A2
L10'	lox71 [O16+O17] (0.2µM)	2	D22.1 (pSB1A2 Eco, Pst)	2 (+6 H2O)	lox71 cloned as a biobrick