Team:Paris/11 August 2009

From 2009.igem.org

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|width=10%| XbaI
|width=10%| XbaI
|width=10%| PstI
|width=10%| PstI
 +
|width=10%| Total
|- style="background: #E0E9EF; text-align: center;"
|- style="background: #E0E9EF; text-align: center;"
Line 49: Line 50:
|width=10%| 1µl
|width=10%| 1µl
|width=10%| 1µl
|width=10%| 1µl
 +
|width=10%| 30µl
|}
|}
2h 37°C
2h 37°C
Line 58: Line 60:
</div>
</div>
<div class="experience">
<div class="experience">
 +
[https://2009.igem.org/Team:Paris/Freezer_PCR_products#A11 A11] (OmpAsignal) and [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A13 A13] (TolRII) after digestion
 +
promega kits
promega kits
50µl  
50µl  
 +
 +
now A11=[https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D13 D13] A13=[https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D14 D14]
</div>
</div>
 +
<div class="caroline">
 +
Ligation
 +
</div>
 +
<div class="experience">
 +
[https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D13 D13] (OmpAsignal X/P) and [https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D14 D14] (TolRII X/P) and vecteur [https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#P16 pSB1A3] digested with XbaI and PstI
 +
 +
DO : D13=0,94  D14=1,29  pSB1A3=0,74
 +
 +
{|
 +
|- style="background: #b3d0E4; text-align: center; color:black; "
 +
|width=10%|
 +
|width=10%|
 +
|width=10%| vecteur
 +
|width=10%| insert
 +
|width=10%| H20
 +
|width=10%| Buffer 10x T4 ligase
 +
|width=10%| T4 ligase
 +
|width=10%| Total
 +
 +
|- style="background: #E0E9EF; text-align: center;"
 +
|width=10%| L3
 +
|width=10%| TolRII:pSB1A3
 +
|width=10%| 2µl
 +
|width=10%| 3µl
 +
|width=10%| 12µl
 +
|width=10%| 2µl
 +
|width=10%| 1µl
 +
|width=10%| 20µl
 +
 +
|- style="background: #E0E9EF; text-align: center;"
 +
|width=10%| L4
 +
|width=10%| OmpAsignal:pSB1A3
 +
|width=10%| 2µl
 +
|width=10%| 3µl
 +
|width=10%| 12µl
 +
|width=10%| 2µl
 +
|width=10%| 1µl
 +
|width=10%| 20µl
 +
 +
|- style="background: #E0E9EF; text-align: center;"
 +
|width=10%| control -
 +
|width=10%| o
 +
|width=10%| 2µl
 +
|width=10%| o
 +
|width=10%| 15µl
 +
|width=10%| 2µl
 +
|width=10%| 1µl
 +
|width=10%| 20µl
 +
|}
 +
 +
 +
1h Home temperature
 +
 +
</div>
 +
<div class="caroline">
 +
Transformation
 +
</div>
 +
<div class="experience">
 +
classic protocol with DH5a
 +
 +
</div>
 +
 +
<div class="charlotte">
 +
Ligation
 +
</div>
 +
<div class="experience">
 +
[https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D11 D11 (RBS-Tet digested by Xba/Pst)] and vecteur [https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D12 D12 (pSB2K3 w/ pLac digested by Spe/Pst)]
 +
 +
DO : D11=  D12= 
 +
 +
{|
 +
|- style="background: #b3d0E4; text-align: center; color:black; "
 +
|width=10%|
 +
|width=10%|
 +
|width=10%| vecteur
 +
|width=10%| insert
 +
|width=10%| H20
 +
|width=10%| Buffer 10x T4 ligase
 +
|width=10%| T4 ligase
 +
|width=10%| Total
 +
 +
|- style="background: #E0E9EF; text-align: center;"
 +
|width=10%| L1
 +
|width=10%| D11
 +
|width=10%| µl
 +
|width=10%| µl
 +
|width=10%| µl
 +
|width=10%| µl
 +
|width=10%| µl
 +
|width=10%| µl
 +
 +
|- style="background: #E0E9EF; text-align: center;"
 +
|width=10%| control -
 +
|width=10%| o
 +
|width=10%| µl
 +
|width=10%| o
 +
|width=10%| µl
 +
|width=10%| µl
 +
|width=10%| µl
 +
|width=10%| µl
 +
|}
 +
</div>
 +
 +
 +
<div class="vicard">
 +
PCR
 +
</div>
 +
<div class="experience">
 +
*For A10(ClyA) because we have 2 bands in the last gel migration of A3(ClyA) purification (06/08/09)
 +
*without DMSO
 +
*with [[https://2009.igem.org/Team:Paris/Freezer_Primers#Freezer 010]] and [[https://2009.igem.org/Team:Paris/Freezer_Primers#Freezer 031]]
 +
** 1:98°C 1min
 +
** 2:98°C 10s
 +
** 3:55°C, 60°C, and 65°C 30s
 +
** 4:72°C 30s
 +
** 5:72°C 10min
 +
** 6: 4°C ~
 +
 +
*Gel-->[Ladder 1kb/A10 at 55°C/A10 at 60°C/A10 at 65°C]
 +
<center>[[Image:clyA_A10.JPG]]</center>
 +
*A10 at 55°C work the best we have the biggest and brightest band with this temperature.
 +
</div>
 +
 +
<div class="vicard">
 +
Digestion
 +
</div>
 +
<div class="experience">
 +
*Digestion of P4 double terminator with EcoRI/SpeI (D15)in order to ligate with P21 (double terminator)
 +
**20µl of P21
 +
**2µl of EcoR1 and 2µl of SpeI
 +
**0,5µl BSA 100X
 +
**5µl buffer 2 x10
 +
**20,5µl H20
 +
**Vfinal=50µl
 +
**2h at 37°C
 +
 +
*Digestion of P21 (pLac in pSB2K3) with EcoRI/XbaI (D16)in order to ligate with P4 (double terminator)
 +
**20µl of P21
 +
**2µl of EcoR1 and 2µl of Xba1
 +
**0,5µl BSA 100X
 +
**5µl buffer 2 x10
 +
**20,5µl H20
 +
**Vfinal=50µl
 +
**2h at 37°C
 +
 +
*Gel-->[Ladder 100pb/D15/Ladder 1kb/D16]
 +
*(15min migration)<center>[[Image:Digestion_1.JPG]]</center>
 +
*(35min migration)<center>[[Image:Digestion_2.JPG]]</center>
 +
</div>
 +
 +
<div class="guillaume">
 +
All Manip
 +
</div>
 +
<div class="experience">
 +
Comming soon
 +
</div>
<html>
<html>

Latest revision as of 22:38, 16 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 11th

Brain work

edit please ^^


Lab work

Digestion

A11 (OmpAsignal) and A13 (TolRII)

DNA Buffer 10X H20 BSA XbaI PstI Total
20µl 3µl 4,5µl 0,5µl 1µl 1µl 30µl

2h 37°C

20min 65°C (stop enzymes)

purification

A11 (OmpAsignal) and A13 (TolRII) after digestion

promega kits

50µl

now A11=D13 A13=D14

Ligation

D13 (OmpAsignal X/P) and D14 (TolRII X/P) and vecteur pSB1A3 digested with XbaI and PstI

DO : D13=0,94 D14=1,29 pSB1A3=0,74

vecteur insert H20 Buffer 10x T4 ligase T4 ligase Total
L3 TolRII:pSB1A3 2µl 3µl 12µl 2µl 1µl 20µl
L4 OmpAsignal:pSB1A3 2µl 3µl 12µl 2µl 1µl 20µl
control - o 2µl o 15µl 2µl 1µl 20µl


1h Home temperature

Transformation

classic protocol with DH5a

Ligation

D11 (RBS-Tet digested by Xba/Pst) and vecteur D12 (pSB2K3 w/ pLac digested by Spe/Pst)

DO : D11= D12=

vecteur insert H20 Buffer 10x T4 ligase T4 ligase Total
L1 D11 µl µl µl µl µl µl
control - o µl o µl µl µl µl


PCR

  • For A10(ClyA) because we have 2 bands in the last gel migration of A3(ClyA) purification (06/08/09)
  • without DMSO
  • with [010] and [031]
    • 1:98°C 1min
    • 2:98°C 10s
    • 3:55°C, 60°C, and 65°C 30s
    • 4:72°C 30s
    • 5:72°C 10min
    • 6: 4°C ~
  • Gel-->[Ladder 1kb/A10 at 55°C/A10 at 60°C/A10 at 65°C]
ClyA A10.JPG
  • A10 at 55°C work the best we have the biggest and brightest band with this temperature.

Digestion

  • Digestion of P4 double terminator with EcoRI/SpeI (D15)in order to ligate with P21 (double terminator)
    • 20µl of P21
    • 2µl of EcoR1 and 2µl of SpeI
    • 0,5µl BSA 100X
    • 5µl buffer 2 x10
    • 20,5µl H20
    • Vfinal=50µl
    • 2h at 37°C
  • Digestion of P21 (pLac in pSB2K3) with EcoRI/XbaI (D16)in order to ligate with P4 (double terminator)
    • 20µl of P21
    • 2µl of EcoR1 and 2µl of Xba1
    • 0,5µl BSA 100X
    • 5µl buffer 2 x10
    • 20,5µl H20
    • Vfinal=50µl
    • 2h at 37°C
  • Gel-->[Ladder 100pb/D15/Ladder 1kb/D16]
  • (15min migration)
    Digestion 1.JPG
  • (35min migration)
    Digestion 2.JPG

All Manip

Comming soon

To do list

Matricule TODO
Luc
Romain
Charlotte
Stoff
Chris
Lisa
Caroline
Souf
Vicard
Pierre
Sylvain
Guillaume


← Yesterday - Tomorrow →

Retrieved from "http://2009.igem.org/Team:Paris/11_August_2009"