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 [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A8 A8] and [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A9 A9]
+
*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
 +
*Duplicate with and without DMSO
**1:98°C 1min
**1:98°C 1min
-
**2:98°C 10s (Not sure to modify guys!)
+
**2:98°C 10s
-
**3:59°C (for A8) /65°C (for A9) 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
Line 49: Line 49:
<div class="guillaume">
<div class="guillaume">
-
Do
+
Do for sequencing
</div>
</div>
<div class="experience">
<div class="experience">
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|width=10%| D0(320)
|width=10%| D0(320)
|width=10%| Dilution Rule
|width=10%| Dilution Rule
 +
|width=10%| Primer Addition
|width=10%| Water Addition  
|width=10%| Water Addition  
Line 69: Line 70:
|width=10%| 0.004
|width=10%| 0.004
|width=10%| 600/310 = 1.93 ~ 2.0 µL
|width=10%| 600/310 = 1.93 ~ 2.0 µL
-
|width=10%| 2.45/10.55 ~ 13 µL  
+
|width=10%| 2.45 µL
 +
|width=10%| 10.55 µL  
|- style="background: #E0E9EF; text-align: center;"
|- style="background: #E0E9EF; text-align: center;"
Line 77: Line 79:
|width=10%| 0.000
|width=10%| 0.000
|width=10%| 600/134 = 4.48 (~ 4.5 µL)
|width=10%| 600/134 = 4.48 (~ 4.5 µL)
-
|width=10%| 2.45/8.05 ~ 10.5 µL
+
|width=10%| 2.45 µL
 +
|width=10%| 8.05 µL
|- style="background: #E0E9EF; text-align: center;"
|- style="background: #E0E9EF; text-align: center;"
Line 85: Line 88:
|width=10%| 0.000
|width=10%| 0.000
|width=10%| 600/70  = 8.57 ~ 8.6 µL
|width=10%| 600/70  = 8.57 ~ 8.6 µL
-
|width=10%| 2.45/3.95 ~ 6.4 µL
+
|width=10%| 2.45µL
-
 
+
|width=10%| 3.95µL
|}
|}
-
 
</div>
</div>
-
<div class="Guillaume">
+
<div class="guillaume">
Gel Migration
Gel Migration
</div>
</div>
<div class="experience">
<div class="experience">
 +
GEL!!!
 +
*Remigrate to be sure... pFecA seem to be good but not Ompa-Linker
 +
</div>
-
*legend to edit....
+
<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>
-
<center>[[Image:KR000634.JPG]][[Image:A3_cut.JPG]]</center>
+
<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>
-
*legend to edit
+
<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>
-
<center>[[Image:KR000637.JPG]][[Image:A5_coupe.JPG]]</center>
+
<div class="caroline">
 +
PCR
</div>
</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>
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|- 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
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8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
MTWTFSS
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6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 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"