Team:Paris/5 August 2009

From 2009.igem.org

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==Brain work==
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===Lab work===
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edit...
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====Microscope====
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<div id="paris_content_boxtop">
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==Lab work==
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{|
{|
|- style="background: #7B96B3; "
|- style="background: #7B96B3; "
-
! colspan="11" style="background: #7B96B3;border:1px solid #333333 "|FM4-64 dying (wash then stain) strain MG4
+
! colspan="11" style="background: #B40ECD;border:1px solid #333333 "|FM4-64 dying (wash then stain) strain MG4
|- style="background: #b3d0E4; text-align: center; color:black; "
|- style="background: #b3d0E4; text-align: center; color:black; "
|width=10%| n°1
|width=10%| n°1
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|width=10%| -
|width=10%| -
-
|- style="background: #f86d6d; text-align: center;"
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|- style="background: #cc6666; text-align: center;"
|width=10%| nothing
|width=10%| nothing
|width=10%| all cell fluo
|width=10%| all cell fluo
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|width=10%| so much dye
|width=10%| so much dye
|}
|}
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{|
{|
|- style="background: #7B96B3; "
|- style="background: #7B96B3; "
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! colspan="11" style="background: #7B96B3;border:1px solid #333333 "|FM4-64 dying (stain then wash)
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! colspan="11" style="background: #B40ECD;border:1px solid #333333 "|FM4-64 dying (stain then wash)
|- style="background: #b3d0E4; text-align: center; color:black; "
|- style="background: #b3d0E4; text-align: center; color:black; "
|width=10%| n°5
|width=10%| n°5
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|- style="background: #E0E9EF; text-align: center;"
|- style="background: #E0E9EF; text-align: center;"
|width=10%| centri 4000rpm 3min
|width=10%| centri 4000rpm 3min
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|width=10%| -
 +
|width=10%| -
|width=10%| -
|width=10%| -
|width=10%| -
|width=10%| -
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|width=10%| + filtrage  
|width=10%| + filtrage  
|width=10%| + filtrage
|width=10%| + filtrage
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|width=10%| -
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|width=10%| -
|- style="background: #E0E9EF; text-align: center;"
|- style="background: #E0E9EF; text-align: center;"
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|width=10%| -
|width=10%| -
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|- style="background: #f86d6d; text-align: center;"
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|- style="background: #cc6666; text-align: center;"
|width=10%| nothing
|width=10%| nothing
|width=10%| some cell fluo
|width=10%| some cell fluo
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|width=10%| noise
|width=10%| noise
|}
|}
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<br>
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<div style="background-color:#7B96B3; border:1px solid #333333; font-weight:bold; padding:3px 5px; margin-bottom:1px">
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<div class="caroline">
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FM4-64 dying with Kayo  
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FM4-64 dying with Kayo
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</div><div style="background-color:#E0E9EF; border:1px solid #333333; padding:3px 5px; margin-bottom:10px">
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</div>
 +
<div class="experience">
protocol like n°2 = all cell fluo, no vesicle
protocol like n°2 = all cell fluo, no vesicle
 +
protocol like n°6 = some cell fluo, noise, no vesicle  
protocol like n°6 = some cell fluo, noise, no vesicle  
</div>
</div>
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<br>
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<br>
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====Molecular biology====
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<div style="background-color:#7B96B3; border:1px solid #333333; font-weight:bold; padding:3px 5px; margin-bottom:1px">
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<div class="guillaume">
Glycerol Stock
Glycerol Stock
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</div><div style="background-color:#E0E9EF; border:1px solid #333333; padding:3px 5px; margin-bottom:10px">
 
-
*[S20] JW4251, [S21] JN0729, [S22] JN0728, [S23] JN0727, [S24] JN0940, [S24] JN0940, [S25] JW5100, [S25] JW5100, [S26] JN5181, [S27] FF64, [S28] NEC280.
 
</div>
</div>
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<div style="background-color:#7B96B3; border:1px solid #333333; font-weight:bold; padding:3px 5px; margin-bottom:1px">
+
<div class="experience">
 +
*[https://2009.igem.org/Team:Paris/Freezer_Strains_(Glycerol_stock)#S20 S20] JW4251, [https://2009.igem.org/Team:Paris/Freezer_Strains_(Glycerol_stock)#S21 S21] JN0729, [https://2009.igem.org/Team:Paris/Freezer_Strains_(Glycerol_stock)#S22 S22] JN0728, [https://2009.igem.org/Team:Paris/Freezer_Strains_(Glycerol_stock)#S23 S23] JN0727, [https://2009.igem.org/Team:Paris/Freezer_Strains_(Glycerol_stock)#S24 S24] JN0940, S24 JN0940, [https://2009.igem.org/Team:Paris/Freezer_Strains_(Glycerol_stock)#S25 S25] JW5100, S25 JW5100, [https://2009.igem.org/Team:Paris/Freezer_Strains_(Glycerol_stock)#S26 S26] JN5181, [https://2009.igem.org/Team:Paris/Freezer_Strains_(Glycerol_stock)#S27 S27] FF64, [https://2009.igem.org/Team:Paris/Freezer_Strains_(Glycerol_stock)#S28 S28] NEC280.
 +
</div>
 +
 
 +
<div class="guillaume">
Miniprep
Miniprep
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</div><div style="background-color:#E0E9EF; border:1px solid #333333; padding:3px 5px; margin-bottom:10px">
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</div>
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<div class="experience">
*Miniprep for P1/2/3/4/5/7/8
*Miniprep for P1/2/3/4/5/7/8
*Same protocol as yesterday. Storage at -20°C.
*Same protocol as yesterday. Storage at -20°C.
</div>
</div>
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<div style="background-color:#7B96B3; border:1px solid #333333; font-weight:bold; padding:3px 5px; margin-bottom:1px">
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 +
<div class="vicard">
Purification
Purification
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</div><div style="background-color:#E0E9EF; border:1px solid #333333; padding:3px 5px; margin-bottom:10px">
 
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*Purification (using 1%agarose at 30min migration) of P2 BBa-J61002 with  XbaI PstI (put pTet forward). The digestion product is 2053pb
 
</div>
</div>
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<div style="background-color:#7B96B3; border:1px solid #333333; font-weight:bold; padding:3px 5px; margin-bottom:1px">
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<div class="experience">
 +
*Purification (using 1%agarose at 30min migration) of [https://2009.igem.org/Team:Paris/Freezer_Plasmids#P2 P2]=BBa-J61002 with  XbaI PstI (put pTet forward).
 +
*D6=2053pb=[https://2009.igem.org/Team:Paris/Freezer_Plasmids#P2 P2] Digested
 +
*[https://2009.igem.org/Team:Paris/Freezer_Plasmids#P2 P2]/nothing/ladder 100pb]
 +
 
 +
 
 +
<center>[[Image:GelP2_o.JPG]][[Image:GelP2_coupé_o.JPG]]</center>
 +
 
 +
 
 +
*P2 purification checking with 1%agarose at 35 min at 75mV
 +
*P2
 +
 
 +
 
 +
<center>[[Image:Gel_apres_purif_o.JPG]]</center>
 +
 
 +
 
 +
*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.
 +
</div>
 +
<span/ id="PCR">
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<div class="guillaume">
PCR
PCR
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</div><div style="background-color:#E0E9EF; border:1px solid #333333; padding:3px 5px; margin-bottom:10px">
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</div>
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<div class="experience">
*Dilution oligo 1/10 (100µM to 10µM)
*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]]
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**2µl Genomic DNA of K12 MG1655 &Delta;recA::Kan
**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)
*Mix x1 Vf=50µl in PCR tube (in ice) (Add with this order)
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**4°C, -
**4°C, -
</div>
</div>
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<div style="background-color:#7B96B3; border:1px solid #333333; font-weight:bold; padding:3px 5px; margin-bottom:1px">
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<div class="guillaume">
Gel migration
Gel migration
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</div><div style="background-color:#E0E9EF; border:1px solid #333333; padding:3px 5px; margin-bottom:10px">
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</div>
 +
<div class="experience">
*Gel 1%: 5µl of [A1|A2|100bp|A3|A4|1kb|A5|A6]
*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-)
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**[https://2009.igem.org/Team:Paris/Freezer_PCR_products#A1 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-)
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**[https://2009.igem.org/Team:Paris/Freezer_PCR_products#A2 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
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**[https://2009.igem.org/Team:Paris/Freezer_PCR_products#A3 A3] = Amplification of clyA with RBS in the primer = 987bp
-
**A4 = Amplification of OmpA signal for export to periplasm = 135bp
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**[https://2009.igem.org/Team:Paris/Freezer_PCR_products#A4 A4] = Amplification of OmpA signal for export to periplasm = 135bp
-
**A5 = Amplification of tolR = 279bp (Try again because of the second band)
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**[https://2009.igem.org/Team:Paris/Freezer_PCR_products#A5 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)
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**[https://2009.igem.org/Team:Paris/Freezer_PCR_products#A6 A6] = Amplification of tatA with RBS = 1634bp (Try again because of the second band)
 +
 
 +
 
<center>[[Image:100bp.png]][[Image:1kb.gif]][[Image:PCR_050809.png]]</center>
<center>[[Image:100bp.png]][[Image:1kb.gif]][[Image:PCR_050809.png]]</center>
</div>
</div>
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<div style="background-color:#7B96B3; border:1px solid #333333; font-weight:bold; padding:3px 5px; margin-bottom:1px">
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<div class="guillaume">
ON culture
ON culture
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</div><div style="background-color:#E0E9EF; border:1px solid #333333; padding:3px 5px; margin-bottom:10px">
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</div>
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*[S8] DH5α(pSB2K3): LB Kan
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<div class="experience">
-
*[29] DH5α(pSB1A3): LB Amp
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*[https://2009.igem.org/Team:Paris/Freezer_Strains_(Glycerol_stock)#S8 S8] DH5α(pSB2K3): LB Kan
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*[30] S113 iGEM08 = DH5α(BBa_K136050): LB Amp
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*[https://2009.igem.org/Team:Paris/Freezer_Strains_(Glycerol_stock)#S29 S29] DH5α(pSB1A3): LB Amp
 +
*[https://2009.igem.org/Team:Paris/Freezer_Strains_(Glycerol_stock)#S30 S30] S113 iGEM08 = DH5α(BBa_K136050): LB Amp
</div>
</div>
<html>
<html>
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==To do list==
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===To do list===
{|
{|
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|Miniprep for P(1/2/3/4/5/7/8) / New P7 digestion? / Glycerol Stock (DH5&alpha;/Kaio strains)
|Miniprep for P(1/2/3/4/5/7/8) / New P7 digestion? / Glycerol Stock (DH5&alpha;/Kaio strains)
|}
|}
 +
 +
 +
{{Paris2009_Calendar_Link|4_August_2009|6_August_2009}}

Latest revision as of 13:46, 10 August 2009

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]

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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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