Team:Paris/10 August 2009

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NoteBook

June
M	T	W	T	F	S	S
[http://2009.igem.org/Team:Paris/1_June_2009 1]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/2_June_2009&action=edit 2]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/3_June_2009&action=edit 3]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/4_June_2009&action=edit 4]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/5_June_2009&action=edit 5]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/6_June_2009&action=edit 6]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/7_June_2009&action=edit 7]
[http://2009.igem.org/wiki/index.php?title=Team:Paris/8_June_2009&action=edit 8]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/9_June_2009&action=edit 9]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/10_June_2009&action=edit 10]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/11_June_2009&action=edit 11]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/12_June_2009&action=edit 12]	[http://2009.igem.org/Team:Paris/13_June_2009 13]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/14_June_2009&action=edit 14]
[http://2009.igem.org/wiki/index.php?title=Team:Paris/15_June_2009&action=edit 15]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/16_June_2009&action=edit 16]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/17_June_2009&action=edit 17]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/18_June_2009&action=edit 18]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/19_June_2009&action=edit 19]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/20_June_2009&action=edit 20]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/21_June_2009&action=edit 21]
[http://2009.igem.org/wiki/index.php?title=Team:Paris/22_June_2009&action=edit 22]	[http://2009.igem.org/Team:Paris/23_June_2009 23]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/24_June_2009&action=edit 24]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/25_June_2009&action=edit 25]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/26_June_2009&action=edit 26]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/27_June_2009&action=edit 27]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/28_June_2009&action=edit 28]
[http://2009.igem.org/wiki/index.php?title=Team:Paris/29_June_2009&action=edit 29]	[http://2009.igem.org/Team:Paris/30_June_2009 30]

July
M	T	W	T	F	S	S
		[http://2009.igem.org/wiki/index.php?title=Team:Paris/1_July_2009&action=edit 1]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/2_July_2009&action=edit 2]	[http://2009.igem.org/Team:Paris/3_July_2009 3]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/4_July_2009&action=edit 4]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/5_July_2009&action=edit 5]
[http://2009.igem.org/wiki/index.php?title=Team:Paris/6_July_2009&action=edit 6]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/7_July_2009&action=edit 7]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/8_July_2009&action=edit 8]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/9_July_2009&action=edit 9]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/10_July_2009&action=edit 10]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/11_July_2009&action=edit 11]	[http://2009.igem.org/wiki/index.php?title=Team:Paris/12_July_2009&action=edit 12]
[http://2009.igem.org/Team:Paris/13_July_2009 13]	[http://2009.igem.org/Team:Paris/14_July_2009 14]	[http://2009.igem.org/Team:Paris/15_July_2009 15]	[http://2009.igem.org/Team:Paris/16_July_2009 16]	[http://2009.igem.org/Team:Paris/17_July_2009 17]	[http://2009.igem.org/Team:Paris/18_July_2009 18]	[http://2009.igem.org/Team:Paris/19_July_2009 19]
[http://2009.igem.org/Team:Paris/20_July_2009 20]	[http://2009.igem.org/Team:Paris/21_July_2009 21]	[http://2009.igem.org/Team:Paris/22_July_2009 22]	[http://2009.igem.org/Team:Paris/23_July_2009 23]	[http://2009.igem.org/Team:Paris/24_July_2009 24]	[http://2009.igem.org/Team:Paris/25_July_2009 25]	[http://2009.igem.org/Team:Paris/26_July_2009 26]
[http://2009.igem.org/Team:Paris/27_July_2009 27]	[http://2009.igem.org/Team:Paris/28_July_2009 28]	[http://2009.igem.org/Team:Paris/29_July_2009 29]	[http://2009.igem.org/Team:Paris/30_July_2009 30]	[http://2009.igem.org/Team:Paris/31_July_2009 31]

August
M	T	W	T	F	S	S
					[http://2009.igem.org/Team:Paris/1_August_2009 1]	[http://2009.igem.org/Team:Paris/2_August_2009 2]
[http://2009.igem.org/Team:Paris/3_August_2009 3]	[http://2009.igem.org/Team:Paris/4_August_2009 4]	[http://2009.igem.org/Team:Paris/5_August_2009 5]	[http://2009.igem.org/Team:Paris/6_August_2009 6]	[http://2009.igem.org/Team:Paris/7_August_2009 7]	[http://2009.igem.org/Team:Paris/8_August_2009 8]	[http://2009.igem.org/Team:Paris/9_August_2009 9]
[http://2009.igem.org/Team:Paris/10_August_2009 10]	[http://2009.igem.org/Team:Paris/11_August_2009 11]	[http://2009.igem.org/Team:Paris/12_August_2009 12]	[http://2009.igem.org/Team:Paris/13_August_2009 13]	[http://2009.igem.org/Team:Paris/14_August_2009 14]	[http://2009.igem.org/Team:Paris/15_August_2009 15]	[http://2009.igem.org/Team:Paris/16_August_2009 16]
[http://2009.igem.org/Team:Paris/17_August_2009 17]	[http://2009.igem.org/Team:Paris/18_August_2009 18]	[http://2009.igem.org/Team:Paris/19_August_2009 19]	[http://2009.igem.org/Team:Paris/20_August_2009 20]	[http://2009.igem.org/Team:Paris/21_August_2009 21]	[http://2009.igem.org/Team:Paris/22_August_2009 22]	[http://2009.igem.org/Team:Paris/23_August_2009 23]
[http://2009.igem.org/Team:Paris/24_August_2009 24]	[http://2009.igem.org/Team:Paris/25_August_2009 25]	[http://2009.igem.org/Team:Paris/26_August_2009 26]	[http://2009.igem.org/Team:Paris/27_August_2009 27]	[http://2009.igem.org/Team:Paris/28_August_2009 28]	[http://2009.igem.org/Team:Paris/29_August_2009 29]	[http://2009.igem.org/Team:Paris/30_August_2009 30]
[http://2009.igem.org/Team:Paris/31_August_2009 31]

September
M	T	W	T	F	S	S
	[http://2009.igem.org/Team:Paris/1_September_2009 1]	[http://2009.igem.org/Team:Paris/2_September_2009 2]	[http://2009.igem.org/Team:Paris/3_September_2009 3]	[http://2009.igem.org/Team:Paris/4_September_2009 4]	[http://2009.igem.org/Team:Paris/5_September_2009 5]	[http://2009.igem.org/Team:Paris/6_September_2009 6]
[http://2009.igem.org/Team:Paris/7_September_2009 7]	[http://2009.igem.org/Team:Paris/8_September_2009 8]	[http://2009.igem.org/Team:Paris/9_September_2009 9]	[http://2009.igem.org/Team:Paris/10_September_2009 10]	[http://2009.igem.org/Team:Paris/11_September_2009 11]	[http://2009.igem.org/Team:Paris/12_September_2009 12]	[http://2009.igem.org/Team:Paris/13_September_2009 13]
[http://2009.igem.org/Team:Paris/14_September_2009 14]	[http://2009.igem.org/Team:Paris/15_September_2009 15]	[http://2009.igem.org/Team:Paris/16_September_2009 16]	[http://2009.igem.org/Team:Paris/17_September_2009 17]	[http://2009.igem.org/Team:Paris/18_September_2009 18]	[http://2009.igem.org/Team:Paris/19_September_2009 19]	[http://2009.igem.org/Team:Paris/20_September_2009 20]
[http://2009.igem.org/Team:Paris/21_September_2009 21]	[http://2009.igem.org/Team:Paris/22_September_2009 22]	[http://2009.igem.org/Team:Paris/23_September_2009 23]	[http://2009.igem.org/Team:Paris/24_September_2009 24]	[http://2009.igem.org/Team:Paris/25_September_2009 25]	[http://2009.igem.org/Team:Paris/26_September_2009 26]	[http://2009.igem.org/Team:Paris/27_September_2009 27]
[http://2009.igem.org/Team:Paris/28_September_2009 28]	[http://2009.igem.org/Team:Paris/29_September_2009 29]	[http://2009.igem.org/Team:Paris/30_September_2009 30]

October
M	T	W	T	F	S	S
			[http://2009.igem.org/Team:Paris/1_October_2009 1]	[http://2009.igem.org/Team:Paris/2_October_2009 2]	[http://2009.igem.org/Team:Paris/3_October_2009 3]	[http://2009.igem.org/Team:Paris/4_October_2009 4]
[http://2009.igem.org/Team:Paris/5_October_2009 5]	[http://2009.igem.org/Team:Paris/6_October_2009 6]	[http://2009.igem.org/Team:Paris/7_October_2009 7]	[http://2009.igem.org/Team:Paris/8_October_2009 8]	[http://2009.igem.org/Team:Paris/9_October_2009 9]	[http://2009.igem.org/Team:Paris/10_October_2009 10]	[http://2009.igem.org/Team:Paris/11_October_2009 11]
[http://2009.igem.org/Team:Paris/12_October_2009 12]	[http://2009.igem.org/Team:Paris/13_October_2009 13]	[http://2009.igem.org/Team:Paris/14_October_2009 14]	[http://2009.igem.org/Team:Paris/15_October_2009 15]	[http://2009.igem.org/Team:Paris/16_October_2009 16]	[http://2009.igem.org/Team:Paris/17_October_2009 17]	[http://2009.igem.org/Team:Paris/18_October_2009 18]
[http://2009.igem.org/Team:Paris/19_October_2009 19]	[http://2009.igem.org/Team:Paris/20_October_2009 20]	[http://2009.igem.org/Team:Paris/21_October_2009 21]	[http://2009.igem.org/Team:Paris/22_October_2009 22]	[http://2009.igem.org/Team:Paris/23_October_2009 23]	[http://2009.igem.org/Team:Paris/24_October_2009 24]	[http://2009.igem.org/Team:Paris/25_October_2009 25]
[http://2009.igem.org/Team:Paris/26_October_2009 26]	[http://2009.igem.org/Team:Paris/27_October_2009 27]	[http://2009.igem.org/Team:Paris/28_October_2009 28]	[http://2009.igem.org/Team:Paris/29_October_2009 29]	[http://2009.igem.org/Team:Paris/30_October_2009 30]	[http://2009.igem.org/Team:Paris/31_October_2009 31]

← Yesterday - Tomorrow →

August 10th

Brain work

edit please ^^

Lab work

PCR

For A8 OmpA-Linker and A9 pFecA
Duplicate with and without DMSO
- 1:98°C 1min
- 2:98°C 10s
- 3:59°C for A8) / 65°C for A9 30s
- 4:72°C 8s
- 5:72°C 10min
- 6: 4°C ~

Mini-Prep for Plac

Same Protocol as usual ...

Do for sequencing

Dilution for Plasmids 1,2&3
Plasmid	D0(000)	D0(266/280)	D0(320)	Dilution Rule	Primer Addition	Water Addition
P1	3.10	1.72	0.004	600/310 = 1.93 ~ 2.0 µL	2.45 µL	10.55 µL
P2	1.34	1.34	0.000	600/134 = 4.48 (~ 4.5 µL)	2.45 µL	8.05 µL
P3	0.70	3.39	0.000	600/70 = 8.57 ~ 8.6 µL	2.45µL	3.95µL

Gel Migration

GEL!!!

Remigrate to be sure... pFecA seem to be good but not Ompa-Linker

Digestion

Digestion of P21 (pLac in pSB2K3) with SpeI/PstI (D12) in order to insert D11 RBS-tet (XbaI/PstI)
- 20µl of P21
- Vfinal=50µl
- 2h at 37°C

Purification

Promega kit
- 50µl final

GEL!!!(strange! none solution fall as quickly in the wholes)

DO₂₆₀

D11: RBS-tet : 0,10µg/ml ~730bp
D12: pSB2K3(pLac) : 0,96µl/ml ~4400bp

Try for ligation

Ligation between [D12] pSB2K3(pLac) SpeI/PstI and [D11] RBS-tet XbaI/PstI. 20µl final
Ligation 1
- 4,5µl RBS-tet XbaI/PstI
- 1µl pSB2K3(pLac) SpeI/PstI
- 2µl Buffer T4 DNA Lygase 10x
- 1µl T4 DNA Lygase
- 11,5µl H₂₀

Ligation 1
- 10µl RBS-tetR XbaI/PstI
- 1µl pSB2K3(pLac) SpeI/PstI
- 2µl Buffer T4 DNA Lygase 10x
- 1µl T4 DNA Lygase
- 6µl H₂₀

10min at room temperature (~25?)
20min at 80°C

Transformation of the ligation

As usual, but I forget to done transformation with vector only...so I haven't any control... --!

PCR

A11 (OmpAsignal) A12 (Tg3p) A13 (TolRII) without (1,2,3) and with DMSO (4,5,6)

temperature gradient

tube n°1/4 : 2/5 : 3/6

A11 : 68,6 : 70,2 : 71,8

A12 : 62,2 : 62,9 : 64,0

A13 : 71,8 : 73,1 : 74,0

->gel

A11(OmpAsignal) and A13(TolRII) good and nothing for A12(Tg3p)

Purification

purification on tube n°5 (A11.5 : T°: 70,2 with DMSO and A13.5 : T°73,1 with DMSO)with promega kits

50µl final

gel purification -> Very good

Next : Digestion tomorrow

To do list

Matricule	TODO
Luc
Romain
Charlotte
Stoff
Chris
Lisa
Caroline
Souf
Vicard
Pierre	modelisation & Algorithmic (a bit...)
Sylvain
Guillaume