Team:Paris/10 August 2009

From 2009.igem.org

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===Lab work===
===Lab work===
-
 
<div class="guillaume">
<div class="guillaume">
PCR
PCR
</div>
</div>
<div class="experience">
<div class="experience">
-
*For A8 (ex A4) ??name??: Matrix = Genomic DNA of K12, Oligo = O11 and O12
+
*For [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A8 A8] OmpA-Linker and [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A9 A9] pFecA
-
*For A9 (ex A5) ??name??: Matrix = Genomic DNA of K12, Oligo = O1 and O13
+
*Duplicate with and without DMSO
-
**1:98°C 30s
+
**1:98°C 1min
**2:98°C 10s
**2:98°C 10s
-
**3:59°C (for A8) /65°C (for A8) 30s   
+
**3:59°C for A8) / 65°C for A9 30s   
**4:72°C  8s
**4:72°C  8s
**5:72°C 10min
**5:72°C 10min
**6: 4°C  ~  
**6: 4°C  ~  
-
NB:1h at 98°C because I haven't push the "enter" buton after it warm up to 98°C...and all evaporate.
 
</div>
</div>
 +
<div class="guillaume">
 +
Mini-Prep for Plac
 +
</div>
 +
<div class="experience">
 +
Same Protocol as usual ...
 +
</div>
 +
<div class="guillaume">
 +
Do for sequencing
 +
</div>
 +
<div class="experience">
 +
{|
 +
|- style="background: #b3d0E4; "
 +
! colspan="11" style="background: #b3d0E4;border:1px solid #333333 "| Dilution for Plasmids 1,2&3
 +
|- style="background: #b3d0E4; text-align: center; color:black; "
 +
|width=10%| Plasmid
 +
|width=10%| D0(000)
 +
|width=10%| D0(266/280)
 +
|width=10%| D0(320)
 +
|width=10%| Dilution Rule
 +
|width=10%| Primer Addition
 +
|width=10%| Water Addition
 +
 +
|- style="background: #E0E9EF; text-align: center;"
 +
|width=10%| [https://2009.igem.org/Team:Paris/Freezer_Plasmids#P1 P1]
 +
|width=10%| 3.10
 +
|width=10%| 1.72
 +
|width=10%| 0.004
 +
|width=10%| 600/310 = 1.93 ~ 2.0 µL
 +
|width=10%| 2.45 µL
 +
|width=10%| 10.55 µL
 +
 +
|- style="background: #E0E9EF; text-align: center;"
 +
|width=10%| [https://2009.igem.org/Team:Paris/Freezer_Plasmids#P2 P2]
 +
|width=10%| 1.34
 +
|width=10%| 1.34
 +
|width=10%| 0.000
 +
|width=10%| 600/134 = 4.48 (~ 4.5 µL)
 +
|width=10%| 2.45 µL
 +
|width=10%| 8.05 µL
 +
 +
|- style="background: #E0E9EF; text-align: center;"
 +
|width=10%| [https://2009.igem.org/Team:Paris/Freezer_Plasmids#P3 P3]
 +
|width=10%| 0.70
 +
|width=10%| 3.39
 +
|width=10%| 0.000
 +
|width=10%| 600/70  = 8.57 ~ 8.6 µL
 +
|width=10%| 2.45µL
 +
|width=10%| 3.95µL
 +
|}
 +
</div>
 +
 +
<div class="guillaume">
 +
Gel Migration
 +
</div>
 +
<div class="experience">
 +
GEL!!!
 +
*Remigrate to be sure... pFecA seem to be good but not Ompa-Linker
 +
</div>
 +
 +
<div class="guillaume">
 +
Digestion
 +
</div>
 +
<div class="experience">
 +
*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
 +
</div>
 +
 +
<div class="guillaume">
 +
Purification
 +
</div>
 +
<div class="experience">
 +
*Promega kit
 +
**50µl final
 +
GEL!!!(strange! none solution fall as quickly in the wholes)
 +
</div>
 +
 +
<div class="guillaume">
 +
DO<sub>260</sub>
 +
</div>
 +
<div class="experience">
 +
*D11: RBS-tet : 0,10µg/ml ~730bp
 +
*D12: pSB2K3(pLac) : 0,96µl/ml ~4400bp
 +
</div>
 +
 +
<div class="guillaume">
 +
Try for ligation
 +
</div>
 +
<div class="experience">
 +
*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 H<sub>20</sub>
 +
<br>
 +
*Ligation 1
 +
**10µl RBS-tetR XbaI/PstI
 +
**1µl pSB2K3(pLac) SpeI/PstI
 +
**2µl Buffer T4 DNA Lygase 10x
 +
**1µl T4 DNA Lygase
 +
**6µl H<sub>20</sub>
 +
<br>
 +
*10min at room temperature (~25?)
 +
*20min at 80°C
 +
</div>
 +
<div class="guillaume">
 +
Transformation of the ligation
 +
</div>
 +
<div class="experience">
 +
*As usual, but I forget to done transformation with vector only...so I haven't any control... --!
 +
</div>
 +
 +
<div class="caroline">
 +
PCR
 +
</div>
 +
<div class="experience">
 +
[https://2009.igem.org/Team:Paris/Freezer_PCR_products#A11 A11] (OmpAsignal) [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A12 A12] (Tg3p) [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A13 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
 +
 +
Waiting Heigh = A11: 135pb, A12: 274pb, A13: 279pb
 +
 +
->gel
 +
 +
[[Image:OmpAsignal_fusion_Nterm.JPG‎]]
 +
 +
 +
[[Image:Tg3p_fusion_N_term.JPG‎]]
 +
 +
 +
[[Image:TolRII fusion N term.JPG‎]]
 +
 +
'''A11(OmpAsignal) and A13(TolRII) good and nothing for A12(Tg3p)'''
 +
 +
</div>
<div class="caroline">
<div class="caroline">
Purification
Purification
</div>
</div>
<div class="experience">
<div class="experience">
-
purification with promega kits
+
purification on tube n°5 (A11.5 : T°: 70,2 with DMSO and A13.5 : T°73,1 with DMSO)with promega kits
-
gel purification -> Very good
+
 
 +
50µl final
 +
 
 +
gel purification -> '''Very good'''
 +
 
 +
[[Image:purif OmpA signal fusion & tolRII fusion N term.JPG‎]]
 +
 
 +
Next : Digestion tomorrow
</div>
</div>
<html>
<html>
Line 93: Line 244:
|- style="background: #e35050;text-align: center;"
|- style="background: #e35050;text-align: center;"
|Pierre
|Pierre
-
|  
+
| modelisation & Algorithmic (a bit...)
|- style="background: #bababa;text-align: center;"
|- style="background: #bababa;text-align: center;"
|Sylvain
|Sylvain

Latest revision as of 11:42, 11 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]

← 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

Waiting Heigh = A11: 135pb, A12: 274pb, A13: 279pb

->gel

OmpAsignal fusion Nterm.JPG


Tg3p fusion N term.JPG


TolRII fusion N term.JPG

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

Purif OmpA signal fusion & tolRII fusion N term.JPG

Next : Digestion tomorrow

To do list

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


← Yesterday - Tomorrow →

Retrieved from "http://2009.igem.org/Team:Paris/10_August_2009"