Team:Paris/5 August 2009

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Contents

NoteBook

June
MTWTFSS
[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
MTWTFSS
    [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
MTWTFSS
          [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
MTWTFSS
  [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
MTWTFSS
      [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 5th

Brain work

edit...

Lab work

FM4-64 dying (wash then stain) strain MG4
n°1 n°2 n°3 n°4
500µl cells - - -
centri 4000rpm 3min - - -
resuspension 500µl MgSO4 - - -
1µl de colorant 1µl de colorant 2µl 2µl
incubation 30min 37°C incubation 60min 37°C incubation 30min 37°C incubation 60min 37°C
depot sur lame - - -
nothing all cell fluo so much dye so much dye
FM4-64 dying (stain then wash)
n°5 n°6 n°9 n°10 n°13 n°14
500µl cells - - - 500µl MgSO4 500µl Medium
1µl de colorant 1µl de colorant 1µl 1µl 1µl 1µl
incubation 30min 37°C incubation 60min 37°C incubation 30min 37°C incubation 60min 37°C incubation 60min 37°C incubation 60min 37°C
centri 4000rpm 3min - - -
resuspension 500µl MgSO4 resuspension 500µl MgSO4 + filtrage + filtrage
depot sur lame - - - - -
nothing some cell fluo noise noise noise noise


FM4-64 dying with Kayo

protocol like n°2 = all cell fluo, no vesicle

protocol like n°6 = some cell fluo, noise, no vesicle



Glycerol Stock

  • [S20] JW4251, [S21] JN0729, [S22] JN0728, [S23] JN0727, [S24] JN0940, [S24] JN0940, [S25] JW5100, [S25] JW5100, [S26] JN5181, [S27] FF64, [S28] NEC280.

Miniprep

  • Miniprep for P1/2/3/4/5/7/8
  • Same protocol as yesterday. Storage at -20°C.

Purification

  • Purification (using 1%agarose at 30min migration) of [P2]=BBa-J61002 with XbaI PstI (put pTet forward).
  • D6=2053pb=[P2]Digested
  • [ladder 1kb/[P2]/nothing/ladder 100pb]
GelP2 o.JPGGelP2 coupé o.JPG
  • [P2] purification checking with 1%agarose at 35 min at 75mV
  • [ladder/nothing/[P2]]
Gel apres purif o.JPG
  • The purification is ok but Ddon't use because it's doesn't have pTet, so we have to cut [P2] with EcorI and SpeI next time.

PCR

  • Dilution oligo 1/10 (100µM to 10µM)
    • 2µl Oligo
    • 18µL H20 RNAse/DNAse free (Gibco)
  • Dilution DNA 1/10
    • 2µl Genomic DNA of K12 MG1655 ΔrecA::Kan [F4]
    • 18µL H20 RNAse/DNAse free (Gibco)
  • Mix x1 Vf=50µl in PCR tube (in ice) (Add with this order)
    • 32,5µl H20 RNAse/DNAse free (Gibco)
    • 10µl Buffer Polymerase fusion 5x
    • 1µl dNTP
    • 2,5µl Oligo F 10µM
    • 2,5µl Oligo R 10µM
    • 1µl Genomic DNA of K12 1/10
    • 0,5µl Polymerase Fusion
  • Mix x6,5
    • 211,25µl H20 RNAse/DNAse free (Gibco)
    • 65µl Buffer Polymerase fusion 5x
    • 6,5µl dNTP
    • 6,5µl Genomic DNA of K12 1/10
    • Put 44,5µl of Mix x6,5 in a PCR tube
    • Add 2,5µl Oligo F 10µM
    • Add 2,5µl Oligo R 10µM
    • Add 0,5µl Polymerase Fusion
  • For A1/2/3/4/5/6, Program PHUSION:
    • 1: 98°C, 1min
    • 2: 98°C, 10sec
    • 3: 65°C, 30sec
    • 4: 72°C, 1min
    • Goto 2 29x
    • 72°C, 10min
    • 4°C, -

Gel migration

  • Gel 1%: 5µl of [A1|A2|100bp|A3|A4|1kb|A5|A6]
    • A1 = Amplification of N-term domain of M13's g3p = 274bp (Useless because we don't use g3p plasmide as matrice => Ctrl-)
    • A2 = Amplification of N-term domain of colicin E3 = 1036bp (Useless because we don't use the right matrice => Ctrl-)
    • A3 = Amplification of clyA with RBS in the primer = 987bp
    • A4 = Amplification of OmpA signal for export to periplasm = 135bp
    • A5 = Amplification of tolR = 279bp (Try again because of the second band)
    • A6 = Amplification of tatA with RBS = 1634bp (Try again because of the second band)
100bp.png1kb.gifPCR 050809.png

ON culture

  • [S8] DH5α(pSB2K3): LB Kan
  • [29] DH5α(pSB1A3): LB Amp
  • [30] S113 iGEM08 = DH5α(BBa_K136050): LB Amp

To do list

Matricule TODO
Luc bacteria coloration microscope protocol
Romain Look for his feet
Charlotte bibliography on SNAREs/Autotransporters/Jun and Fos complex
Stoff merge algo protocol/ Wiki
Chris modeling: TeTR
Lisa WTF !!!
Caroline bacteria coloration microscope protocol
Souf if(!oligo){return Wiki;} return PCR;
Vicard Lab : Gel / purification / insers
Pierre Alice / Tol/Pal modelisation / geophysics
Sylvain if(!oligo){return Maltose;} return PCR;
Guillaume Miniprep for P(1/2/3/4/5/7/8) / New P7 digestion? / Glycerol Stock (DH5α/Kaio strains)


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