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Revision as of 12:41, 20 July 2007 (view source) Landrain (Talk | contribs) (→Glycerol stock) ← Older edit		Latest revision as of 17:49, 7 October 2007 (view source) Nicolas C. (Talk | contribs)
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		+	[[Paris/July 19|yesterday]] -- [[Paris/July 21|tomorrow]] <br>
	==PCRs==		==PCRs==
-
-
-	'''Je n’ai pas pu lancer la PCR car :
-
-	Les oligos O10 et O11 ne sont pas dilués à 10μM (ni les autres oligos après O9 d’ailleurs ??!!). Les fiches des oligos ne sont pas dans le classeur, je ne peux donc pas savoir à quelle concentration est le stock !'''
-	NB : Tous les oligos en stock sont à 100µM. Et ils sont ensuite dilués à 10µM dans l'autre boïte
-
	{{Paris_PCR_0| Title = Lox71-KmR-Lox66		{{Paris_PCR_0| Title = Lox71-KmR-Lox66
-	|Annealing= 55°(3x)-63°(35x)C	+	|Annealing= 55
-	|Elongation= 2m00'	+	|Elongation= 3m00'
-	|Cycles= 38	+	|Cycles= 35
	|Buffer=  5x 10µL		|Buffer=  5x 10µL
	|MgCl2= 10µM 0µL		|MgCl2= 10µM 0µL
Line 19:		Line 13:
	|n_oligoR= 11  Lox66-KmR-R		|n_oligoR= 11  Lox66-KmR-R
	|v_oligoR= 2.5µL		|v_oligoR= 2.5µL
-	|water= 30µL	+	|water= 34µl
	|pol= Phusion 0.5µL		|pol= Phusion 0.5µL
-	|DNA= MP3_pSB1AK3-BBa_B0015 2µL dil.100	+	|DNA= MP3_pSB1AK3-BBa_B0015 0.5µl
-	|Size=	+	|Size= 1084
-	|Success=	+	|Success= ?
		+	|Image=
		+	|Band=
		+	|}}
		+
		+	{{Paris_PCR_0| Title = Lox71-FtsA-FtsZ
		+	|Annealing= 55
		+	|Elongation= 3m00'
		+	|Cycles= 35
		+	|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= toothpick in MG1655 glycerol
		+	|Size= 2588
		+	|Success= ? The PCR seemed to have worked but the purification failed
	|Image=		|Image=
	|Band=		|Band=
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	* BBa_pJ23107 clone 3&4		* BBa_pJ23107 clone 3&4
	* BBa_I0500 clone 1&2		* BBa_I0500 clone 1&2
		+
		+	== E.Coli pKS::DGAT ==
		+
		+	We look under microscopy 36 hours after incubation of E.coli transformed by pKS::DGAT and the control E.coli transformed by part B0015  on different LB medium (See [[Paris/July_18#E.coli_pKS::DGAT|July 18]]).
		+
		+	* Observations:
		+	We can observe colonies (X10).
		+	[[Image: coli_dgat_07202007.jpg|center|900px]]
		+
		+	* Interpretation
		+
		+	We can observe lipid inclusions within colonies with DGAT. Further investigations need single cell visualisation.

---

Latest revision as of 17:49, 7 October 2007

yesterday -- tomorrow

Contents 1 PCRs 2 Glycerol stock 3 Minipreps on Biobricks 4 E.Coli pKS::DGAT

PCRs

PCR : Lox71-KmR-Lox66
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL			1084
55		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	10 Lox71-KmR-F	2.5µL		?
3m00'		Oligo R 10µM	11 Lox66-KmR-R	2.5µL		Image (click to enlarge)
Number cycles		Water	34µl			[[Image:|30px]]
35		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	MP3_pSB1AK3-BBa_B0015 0.5µl

PCR : Lox71-FtsA-FtsZ
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL			2588
55		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	3 Lox71-FtsA-F	2.5µL		? The PCR seemed to have worked but the purification failed
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 MG1655 glycerol

Glycerol stock

• BBa_pJ23107 clone 3&4 (S14)
• BBa_I0500 clone 1&2 (S13)

Minipreps on Biobricks

• BBa_pJ23107 clone 3&4
• BBa_I0500 clone 1&2

E.Coli pKS::DGAT

We look under microscopy 36 hours after incubation of E.coli transformed by pKS::DGAT and the control E.coli transformed by part B0015 on different LB medium (See July 18).

• Observations:

We can observe colonies (X10).

• Interpretation

We can observe lipid inclusions within colonies with DGAT. Further investigations need single cell visualisation.