From 2007.igem.org

Revision as of 16:22, 16 August 2007 (view source) David.bikard (Talk | contribs) (→Digestions) ← Older edit		Latest revision as of 23:21, 16 August 2007 (view source) Eismoustique (Talk | contribs) (→Ligations)
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Line 113:		Line 113:
	|2		|2
	|control of ligation efficiency		|control of ligation efficiency
-	|	+	|~1000
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |C2		| style="background: #ccffcc;" |C2
Line 121:		Line 121:
	|2µl		|2µl
	|control of D35 digestion		|control of D35 digestion
-	|	+	|5
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |C4		| style="background: #ccffcc;" |C4
	|		|
	|		|
-	|D? (L9 lox66 EX)	+	|D42 (L9 lox66 EX)
	|2µl		|2µl
-	|control of D? digestion	+	|control of D42 digestion
-	|	+	|5
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |L26		| style="background: #ccffcc;" |L26
	|D33.2 (L6.2 RBS-Cre XP)		|D33.2 (L6.2 RBS-Cre XP)
	|5		|5
-	|D? (L1 J61002::pbad SP)	+	|D41 (L1 J61002::pbad SP)
	|1		|1
	| Insertion of RBS-Cre behind araC/pBad		| Insertion of RBS-Cre behind araC/pBad
-	|	+	|x100
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |L27		| style="background: #ccffcc;" |L27
	|D27 (ftsZ PCR XP)		|D27 (ftsZ PCR XP)
	|5		|5
-	|D? (L1 J61002::pbad SP)	+	|D41 (L1 J61002::pbad SP)
	|1		|1
	| Insertion of ftsZ behind araC/pBad		| Insertion of ftsZ behind araC/pBad
-	|	+	|6
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |L28		| style="background: #ccffcc;" |L28
	|D32 (ftsAZ PCR XP)		|D32 (ftsAZ PCR XP)
	|5		|5
-	|D? (L1 J61002::pbad SP)	+	|D41 (L1 J61002::pbad SP)
	|1		|1
	| Insertion of ftsAZ behind araC/pBad		| Insertion of ftsAZ behind araC/pBad
-	|	+	|8
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
	| style="background: #ccffcc;" |L29		| style="background: #ccffcc;" |L29
-	|D? (MP8.1 gfp-tripart ES)	+	|D43 (MP8.1 gfp-tripart ES)
	|5		|5
-	|D? (L9 pSB1A2::lox66 XP)	+	|D42 (L9 pSB1A2::lox66 EX)
	|2		|2
	| Insertion of gfp-tripart in front of lox66		| Insertion of gfp-tripart in front of lox66
-	|	+	|x100
	|- style="background: #cccccc;"		|- style="background: #cccccc;"
-	| style="background: #ccffcc;" |L30(.1 & .2)	+	| style="background: #ccffcc;" |L30(A&B)
	|dapAp [O31+O32]0.2µM		|dapAp [O31+O32]0.2µM
	|2		|2
-	|D9.1 & D9.2 (dapAp [O31+O32]0.2µM)	+	|D9.1 & D9.2
	|1		|1
	|cloning dapAp promotor in J61002		|cloning dapAp promotor in J61002
-	|	+	|x100
	|}		|}

---

Latest revision as of 23:21, 16 August 2007

yesterday -- tomorrow

Digestions

Digestion Products
Number	Product Name	Matrix Name	Enzyme 1	Enzyme 2	Size	Description	Product Left	[]ng/µL	Date
D41	araC/pBad-J61002 plasmid BV	L1.2 (araC/pBad-J61002 plasmid)	Spe1	Pst1		Vector with the pBAD promoter ready for insertion of a RBS/CDS insert			August 13
D22	pSB1A2	L6.1 (rbs-CRE)	EcoR1	Pst1					August 13
D33	RBS(B0030)-CreORF BI	L6.2 (rbs-CRE)	XbaI	PstI		RBS-Cre digestion for use as a BI			August 13
D42	Lox66 FV	L9	EcoRI	XbaI		pSB1A2-lox66 as a Forward vector			August 13
D35	Lox71 BV	L10	SpeI	PstI		pSB1A2-lox71 as a Backward vector			August 13
D43	gfp-tripart FI	MP8.1	EcoRI	SpeI		gfp-tri part (strongRBS-ORF-T) ready for use as a forward insert			August 13

digestion mix:

• 25µl plasmid
• 1µl BSA
• 10 µl NEB2
• 60µl H2O
• 2µl Enzyme1
• 2µl Enzyme2

Ligations

Ligations
Number	Insert	Insert Volume (µL)	Vector	Vector Volume (µL)	Comments	Number of colonies
C1			MP3.1 digested with EcoRI (D0)	2	control of ligation efficiency	~1000
C2			D35 (L10 lox71 dig.SP)	2µl	control of D35 digestion	5
C4			D42 (L9 lox66 EX)	2µl	control of D42 digestion	5
L26	D33.2 (L6.2 RBS-Cre XP)	5	D41 (L1 J61002::pbad SP)	1	Insertion of RBS-Cre behind araC/pBad	x100
L27	D27 (ftsZ PCR XP)	5	D41 (L1 J61002::pbad SP)	1	Insertion of ftsZ behind araC/pBad	6
L28	D32 (ftsAZ PCR XP)	5	D41 (L1 J61002::pbad SP)	1	Insertion of ftsAZ behind araC/pBad	8
L29	D43 (MP8.1 gfp-tripart ES)	5	D42 (L9 pSB1A2::lox66 EX)	2	Insertion of gfp-tripart in front of lox66	x100
L30(A&B)	dapAp [O31+O32]0.2µM	2	D9.1 & D9.2	1	cloning dapAp promotor in J61002	x100

Ligation mix:

• 2µl 10x buffer
• 1µl Ligase
• XµL plasmid DNA solution
• YµL insert DNA solution
• H2O qsp 20µL

Reaction in 20µl

ON @ 4°C