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Revision as of 22:56, 23 July 2007 (view source) David.bikard (Talk | contribs) ← Older edit		Revision as of 23:00, 23 July 2007 (view source) David.bikard (Talk | contribs) (→PCRs) Newer edit →
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	|pol= Phusion 0.5µL		|pol= Phusion 0.5µL
	|DNA= Toothpick in MG16655 Glycerol		|DNA= Toothpick in MG16655 Glycerol
		+	|Size= 2058
		+	|Success=
		+	|Image=
		+	|Band=
		+	|}}
		+
		+	{{Paris_PCR_0| Title = Assembly PCR Lox71-FtsA-FtsZ-1 + FtsZ-2
		+	|Annealing= 70°C (5x) without oligos + 60°C (35x)
		+	|Elongation= 3m00'
		+	|Cycles= 40x
		+	|Buffer=  5x 10µL
		+	|MgCl2= 10µM 0µL
		+	|dNTP= 10µM 1µL
		+	|n_oligoF= 3 Lox71-FtsA-F
		+	|v_oligoF= 2.5µL
		+	|n_oligoR= 2 FtsZ-R
		+	|v_oligoR= 2.5µL
		+	|water= 34µl
		+	|pol= Phusion 0.5µL
		+	|DNA= 8.5µl Lox71-FtsA-FtsZ-1 (~50ng) + 17.5µl FtsZ-2 (~50ng)
	|Size= 2058		|Size= 2058
	|Success=		|Success=

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Revision as of 23:00, 23 July 2007

Contents 1 w121 growth 2 Transduction of DapA deletion in MG1655 3 Migration of digestion products 4 MiniPrep 5 Digestion reactions 6 PCRs

w121 growth

w121 culture launched on 22/07/07 did not grow ON: I had forgot to add DAP to LB-Erythro growth medium.

at 12h30, the following culture was launched:

• w121 in 2ml LB-Erythro-DAP

We were unable to perform growth kinetics analysis of w121 in different DAP-supplemented media (because the machine is occupied for the night

Transduction of DapA deletion in MG1655

• The transduction seems to have worked: isolation of clones on LB and LB+DAP for verification.

Migration of digestion products

MiniPrep

• BBa_J23107

Digestion reactions

Each of the digestion reactions that follow were performed as follows:

• 20µL DNA solution (miniprep or PCR product)
• 5µL Buffer 10x
• 0.5µL BSA 100x
• 2µL of each of the 2 enzymes indicated
• H20 qsp 50µL

NEB3 buffer used for XbaI/PstI double digestion NEBEcoRI buffer used for both EcoRI/SpeI & EcoRI/PstI double digestion reactions

Digestion Products
Number	Product Name	Matrix Name	Enzyme 1	Enzyme 2	size	Description
D22	pSB1A2 open vector	BBa_J61047 (MP9.1 & MP9.2)	EcoI	PstI		Open pSB1A2 vector for BioBrick EcoRI/PstI insertio
D23	AraC/pBad promoter fw-insert	BBa_I0500 (MP4.1 & MP4.2)	EcoI	SpeI		AraC/pBad promoter ready for use as a forward insert
D24	lox71-ftsZ bw-insert	lox71-ftsZ PCR product P7	XbaI	PstI		lox71-ftsZ(unmutated) ready for use as a backward insert
D25	eCFP bw-insert	BBa_E0422 (MP6.1 & MP6.2)	XbaI	PstI		eCFP (RBS-OFR_LVA-T) ready for use as a backward insert
D26	PoPs to GFP converter	BBa_E0421 (MP7.1 & MP7.2)	XbaI	PstI		PoPs to GFP converter ready for use as a backward insert
D27	gfp-tripart	BBa_E0840 (MP8.1 & MP8.2)	XbaI	PstI		gfp-tri part (strongRBS-ORF-T) ready for use as a backward insert

PCRs

PCR : Lox71-FtsA-FtsZ
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL			2058
60		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	3 Lox71-FtsA-F	2.5µL
3m00'		Oligo R 10µM	2 FtsZ-R	2.5µL		Image (click to enlarge)
Number cycles		Water	34µl			[[Image:|30px]]
35		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	Toothpick in MG16655 Glycerol

PCR : Assembly PCR Lox71-FtsA-FtsZ-1 + FtsZ-2
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL			2058
70°C (5x) without oligos + 60°C (35x)		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	3 Lox71-FtsA-F	2.5µL
3m00'		Oligo R 10µM	2 FtsZ-R	2.5µL		Image (click to enlarge)
Number cycles		Water	34µl			[[Image:|30px]]
40x		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	8.5µl Lox71-FtsA-FtsZ-1 (~50ng) + 17.5µl FtsZ-2 (~50ng)