Team:Paris/5 August 2009

From 2009.igem.org

Contents

NoteBook

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August
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31
September
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August 5th

Lab work

Microscope

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

Molecular biology

Glycerol Stock

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
  • P2/nothing/ladder 100pb]


GelP2 o.JPGGelP2 coupé o.JPG


  • P2 purification checking with 1%agarose at 35 min at 75mV
  • P2


Gel apres purif o.JPG


  • The purification is ok but don'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
    • 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
  • S29 DH5α(pSB1A3): LB Amp
  • S30 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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