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Revision as of 15:26, 30 July 2007 (view source) David.bikard (Talk | contribs) (→PCRs) ← Older edit		Latest revision as of 17:48, 7 October 2007 (view source) Nicolas C. (Talk | contribs)
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		+	[[Paris/July 16|yesterday]] -- [[Paris/July 18|tomorrow]] <br>
	== PCRs ==		== PCRs ==

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Latest revision as of 17:48, 7 October 2007

yesterday -- tomorrow

Contents 1 PCRs 2 DAP solution contamination test 3 Transduction of MG1655 with P1 stock made on w121 4 Transformations of Biobricks : results 5 Transduction of w121 strain 6 Digestions 7 Exponential culture of E.coli pKS::DGAT and microscopy

PCRs

• The assembly PCRs did not work... we'll try them again !
• The Lox66-DapAColi PCRs worked very well in all conditions

=> Gel purification

PCR : Lox71-FtsZ
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL
55°C		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	1 Lox71-FtsZ-F	10µM 2.5µL		YES
2m30'		Oligo R 10µM	2 FtsZ-R	10µM 2.5µL		Image (click to enlarge)
Number cycles		Water	34µL
30		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	toothpick in glycerol stock of MG1655			1-2

PCR : Lox66-DapA-Subtilis
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL
55°C		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	8 Lox66-DapASubtilis-F	10µM 2.5µL		YES
2m30'		Oligo R 10µM	9 DapASubtilis R	10µM 2.5µL		Image (click to enlarge)
Number cycles		Water	34µL
30		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	0.5µL of Subtilis DNA from stock (DB tube in fridge)			3-4

PCR : Assembly PCR DGAT
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL
55°C		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM		10µM 2.5µL		NO
2m30'		Oligo R 10µM		10µM 2.5µL		Image (click to enlarge)
Number cycles		Water	34µL
30		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	1µL of PCR product DGAT 1 1µL of PCR product DGAT 2			7-8

PCR : Assembly PCR FtsZ-FtsA
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL
55°C		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	3	10µM 2.5µL		NO
2m30'		Oligo R 10µM	2	10µM 2.5µL		Image (click to enlarge)
Number cycles		Water	34µL
30		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	1µL of PCR product FtsZ 1 1µL of PCR product FtsZ 2			5-6

I make a gel to check PCR products. To put on gel :

• 50µL of PCR product
• 5µL of loading buffer.

As the precedent one didn't work, we decided to change our way to do it. We made the PCR reactions within two main steps :

• the 2 PCR products are used as a matrix and added to the PCR mix without the primers ! - then 15 typical cycles
• We add the primers to the PCR mix and run the total for 30 cycles.

We tested the PCR construction. It didn't give anything !

• DGAT = a smear + 1 band at 100kb (primers)
• FtsZ = nothing at all...

Let's do it again tomorow i order to find out where the problem could be from.

DAP solution contamination test

We spread on LB plates DAP solution :

• 50µL of H₂0 (control)
• 50µL of aliquot DAP 50mM (in use)
• 50µL of DAP 1mM
• 50µL of DAP 0.2 mM
• 50µL of DAP 50mM

+ filtration of the DAP stock
See Results.

Transduction of MG1655 with P1 stock made on w121

As it still didn't work, we try for now with culture of MG1655 in exponential growth rate (ExpGR)
OD=0.6

• Preparation of samples:
  • Control (900µL LB MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
  • 5µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
  • 50µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
  • 500µL Phage + 500µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
• 20mn @ 37°C = adsorption
• Centrifugation 1mn @ 10000rpm
• Resuspension in LB + Na Citrate 20mM + DAP 300µM
• 1H growth @ 37°C
• Plate on LB + Na Citrate + Erythromycin + DAP

Transformations of Biobricks : results

Transformation of biobricks : we obtained hundreds of clones for each biobrick. Transformation in DH5alpha subcloning efficiency
Spread on LB-Amp

• BBa_B0030 pSB1A2 : RBS (well 3G plate 1)
• BBa_E0422 pSB1A2 : ECFP (RBS+LVA+Term) (well 11G plate 1)
• BBa_E0241 pSB1A2 : PoPs to GFP converter (well 15c plate 2)
• BBa_E0840 gfp tri-part; strong rbs (well 16E plate 1)
• BBa_J61047 pSB1A2 : Cre ORF (well 8P plate 4)

Transduction of w121 strain

Titration of the stock of phage from 12.7.7.
See results.

Digestions

We did as follow :

• 25µL of plasmid miniprep
  
• 15,5µL H20
  
• 5µL of NE2 Buffer 10X (Biolabs)
  
• 0,5µL of BSA 100X
  
• 2µL of Enzyme1 (Biolabs)
  
• 2µL of Enzyme2 (Biolabs)
  

We have 2 clones for each plasmid, we digested both as follow :

• pJ23100 with EcoR1/Spe1 and Spe1/Pst1
• pJ23107 with EcoR1/Spe1 and Spe1/Pst1
• I0500 with Ecor1/Pst1
• B0015 with EcoR1/Pst1
  
  
• Lox66 DapA E.coli (PCR product) with EcoR1/Pst1 and Xba1/Pst1

After 3 hours of digestion, running on a 1% agarose gel.
We cut the bands and put them all at -20°C for next morning.

Exponential culture of E.coli pKS::DGAT and microscopy

• Protocol

- dilution 1/200 of the liquid culture overnight of E.coli pKS::DGAT and E.coli control (see July 16).

- incubation 2hours 37°C

- add:

1. oleate : 0/0.5mM/2mM
2. IPTG : 0/0.5mM
   

- incubate 1 hour 37°C

- add Nile Red (0.5µg/mL medium)

- microscopy pictures

• Result:

No differences between the different culture condition (+-IPTG / +-oleate / E.coli transformed or not).

We decide to test after overnight culture: no result.