Team:Paris/5 August 2009

From 2009.igem.org

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PCR
PCR
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-
*Dilution oligo 1/10
+
*Dilution oligo 1/10 (100µM to 10µM)
**2µl Oligo
**2µl Oligo
**18µL H<sub>2</sub>0 RNAse/DNAse free (Gibco)
**18µL H<sub>2</sub>0 RNAse/DNAse free (Gibco)
*Dilution DNA 1/10
*Dilution DNA 1/10
-
**2µl Genomic DNA of K12 MG1655 &Delta;recA::Kan [[https://2009.igem.org/Team:Paris/Fridge | F4]]
+
**2µl Genomic DNA of K12 MG1655 &Delta;recA::Kan [[https://2009.igem.org/Team:Paris/Fridge F4]]
**18µL H<sub>2</sub>0 RNAse/DNAse free (Gibco)
**18µL H<sub>2</sub>0 RNAse/DNAse free (Gibco)
-
 
+
*Mix x1 Vf=50µl in PCR tube (in ice) (Add with this order)
-
 
+
**32,5µl H<sub>2</sub>0 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
 +
<br>
 +
*Mix x6,5
 +
**211,25µl H<sub>2</sub>0 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
 +
<br>
 +
*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, -
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Revision as of 19:44, 5 August 2009

Contents

NoteBook

June
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July
MTWTFSS
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August
MTWTFSS
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3 4 5 6 7 8 9
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17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
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October
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← Yesterday - Tomorrow →

August 5th

Brain work

edit...

Lab work

FM4-64 dying (wash then stain)
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



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.

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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