Team:Paris Saclay/Notebook/August/5

From 2013.igem.org

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(→4 - Gel purification of electrophoresis of PCR of PSB1C3)
(→3 - Electrophoresis of PCR of pSB1C3)
 
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* Well 1 : 6µL of DNA Ladder
* Well 1 : 6µL of DNA Ladder
* Well 2 : 30µL of BBa_K1155003 from clone 9 digested by EcoRI/PstI + 6µl of 6X loading dye
* Well 2 : 30µL of BBa_K1155003 from clone 9 digested by EcoRI/PstI + 6µl of 6X loading dye
-
* Well 3 : 5µL of BBa_K1155003 from clone 9 +1µl of 6X loading dye
+
* Well 3 : 5µL of BBa_K1155003 from clone 9 + 1µl of 6X loading dye
* Well 4 : 30µL of BBa_K1155003 from clone 11 digested by EcoRI/PstI + 6µl of 6X loading dye
* Well 4 : 30µL of BBa_K1155003 from clone 11 digested by EcoRI/PstI + 6µl of 6X loading dye
-
* Well 5 : 5µL of BBa_K1155003 from clone 11 +1µl of 6X loading dye
+
* Well 5 : 5µL of BBa_K1155003 from clone 11 + 1µl of 6X loading dye
* Well 6 : 30µL of BBa_K1155003 from clone 12 digested by EcoRI/PstI  +6µl of 6X loading dye
* Well 6 : 30µL of BBa_K1155003 from clone 12 digested by EcoRI/PstI  +6µl of 6X loading dye
-
* Well 7 : 5µL of BBa_K1155003 from clone 12 +1µl of 6X loading dye
+
* Well 7 : 5µL of BBa_K1155003 from clone 12 + 1µl of 6X loading dye
* Gel : 1%
* Gel : 1%
|}  
|}  
Expected sizes :  
Expected sizes :  
-
* RBS_AmilCP-Term :  
+
* RBS_AmilCP-Term : 824bp
* pSB1C3 : 2070bp
* pSB1C3 : 2070bp
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
We obtain fragments at the right size. We will sequence it.  
+
We obtained fragments at the right size. We will sequence it.  
|}
|}
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* SpeI : 2µL
* SpeI : 2µL
-
We incubate the digestion for 1h30 at 37°C.
+
We incubate the digestion at 37°C for 1h30.
===='''2 -Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI'''====  
===='''2 -Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI'''====  
Line 80: Line 80:
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
We obtain fragments at the right size. We will make an elector-elution to extract RBS-LacZ.
+
We obtained fragments at the right size. We will make an electro-elution to extract RBS-LacZ.
|}
|}
-
===='''3 - PCR of PSB1C3'''====
+
===='''3 - PCR of pSB1C3'''====
Nadia, XiaoJing
Nadia, XiaoJing
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* Oligo 65 : 2.5µL
* Oligo 65 : 2.5µL
* dNTP : 1µL
* dNTP : 1µL
-
* Phusion : 0.25µL
+
* enzyme Phusion : 0.25µL
* H2O : 36.75µL
* H2O : 36.75µL
Line 100: Line 100:
Hybridation temperature gradient :  
Hybridation temperature gradient :  
-
A - 65
+
A - 65°C/
-
B - 64.7
+
B - 64.7°C/
-
C - 64.1
+
C - 64.1°C/
-
D - 63.1
+
D - 63.1°C/
-
E - 62
+
E - 62°C/
-
F - 61.2
+
F - 61.2°C/
-
G - 60.5
+
G - 60.5°C/
-
H - 60
+
H - 60°C/
[[File:PsPCRC30508.jpg|400px]]
[[File:PsPCRC30508.jpg|400px]]
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| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
* Well 1 : 6µL of DNA Ladder
* Well 1 : 6µL of DNA Ladder
-
* Well 2 to 9 : 2µL of pSB1C3+3µL of H2O+1µL of 6X loading dye
+
* Well 2 to 9 : 2µL of pSB1C3 + 3µL of H2O + 1µL of 6X loading dye
* Gel : 1%
* Gel : 1%
|}
|}
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{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
We obtain fragments at the right size. We will purify it.
+
We obtained fragments at the right size. We will purify it.
|}
|}
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===='''Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
===='''Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
-
===='''1 - Colony PCR of BBa_K1155004, BBa_K115005, BBa_K1155006 in DH5α'''====
+
===='''1 - PCR Colonies of BBa_K1155004, BBa_K115005, BBa_K1155006 in DH5α'''====
Damir, Nadia, XiaoJing
Damir, Nadia, XiaoJing
Line 148: Line 148:
|}
|}
-
We mix our DNA in 10µL of H2O.
+
We took a single colony and resuspend in 10µL H2O. For each Biobrick, we did 25 PCR Colonies.
Used quantities :  
Used quantities :  
-
* DNA : 2µL
+
 
-
* Mix  : (it was divided in 25 tubes for 25 different colonies for each promotor with 23µL of mix in each tube)
+
PCR preparation mix for 25 different colonies including:
** Oligo 44 : 3.5µL
** Oligo 44 : 3.5µL
** Oligo 45 : 3.5µL
** Oligo 45 : 3.5µL
Line 159: Line 159:
** Dream Taq : 5µL
** Dream Taq : 5µL
** H2O : 590µL   
** H2O : 590µL   
 +
 +
PCR reaction:
 +
* DNA : 2µL of resuspend colony
 +
* Mix PCR  :23µL
 +
Total volume: 25µL
PCR Program :  
PCR Program :  

Latest revision as of 01:25, 5 October 2013

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Contents

Notebook : August 5

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155003

1 - Digestion of BBa_K1155003 by EcoRI/PstI to check the ligation between RBS-Amil CP,Term and pSB1C3

Nadia, XiaoJing

Used quantities :

  • BBa_K1155003 : 5µL
  • EcoRI FD  : 1µL
  • PstI FD : 1µL
  • Buffer FD : 3µL
  • H2O : 20µL

We incubate the digestion at 37°C during 15 minutes.

2 - Electrophoresis of the digestion of BBa_K1155003

Damir

Psgel10508.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 : 30µL of BBa_K1155003 from clone 9 digested by EcoRI/PstI + 6µl of 6X loading dye
  • Well 3 : 5µL of BBa_K1155003 from clone 9 + 1µl of 6X loading dye
  • Well 4 : 30µL of BBa_K1155003 from clone 11 digested by EcoRI/PstI + 6µl of 6X loading dye
  • Well 5 : 5µL of BBa_K1155003 from clone 11 + 1µl of 6X loading dye
  • Well 6 : 30µL of BBa_K1155003 from clone 12 digested by EcoRI/PstI +6µl of 6X loading dye
  • Well 7 : 5µL of BBa_K1155003 from clone 12 + 1µl of 6X loading dye
  • Gel : 1%

Expected sizes :

  • RBS_AmilCP-Term : 824bp
  • pSB1C3 : 2070bp

We obtained fragments at the right size. We will sequence it.

Objective : obtaining BBa_K1155007

1 - Digestion of BBa_I732017 by EcoRI/SpeI

Abdou, Damir, Nadia,

Used quantities :

  • BBa_I732017 : 41µL
  • Buffer FD : 5µL
  • EcoRI : 2µL
  • SpeI : 2µL

We incubate the digestion at 37°C for 1h30.

2 -Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI

Damir, Nadia

Psgel20508.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 : 40µL of BBa_I732017 digested by EcoRI/Spe I+ 8µL 6X loading dye
  • Gel : 1.5%

Expected sizes :

  • RBS-LacZ : 3093 bp
  • pSB1A2 : 2079 bp

We obtained fragments at the right size. We will make an electro-elution to extract RBS-LacZ.

3 - PCR of pSB1C3

Nadia, XiaoJing

Used quantities :

  • pSB1C3 : 2µL
  • Buffer phusion : 10µL
  • Oligo 64 : 2.5µL
  • Oligo 65 : 2.5µL
  • dNTP : 1µL
  • enzyme Phusion : 0.25µL
  • H2O : 36.75µL

PCR program :

Hybridation temperature gradient :

A - 65°C/ B - 64.7°C/ C - 64.1°C/ D - 63.1°C/ E - 62°C/ F - 61.2°C/ G - 60.5°C/ H - 60°C/

PsPCRC30508.jpg

3 - Electrophoresis of PCR of pSB1C3

Nadia, XiaoJing

Psgel30508.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 to 9 : 2µL of pSB1C3 + 3µL of H2O + 1µL of 6X loading dye
  • Gel : 1%

Expected sizes :

  • pSB1C3 : 2070bp

We obtained fragments at the right size. We will purify it.

4 - Gel purification of electrophoresis of PCR of pSB1C3

Nadia, XiaoJing

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - PCR Colonies of BBa_K1155004, BBa_K115005, BBa_K1155006 in DH5α

Damir, Nadia, XiaoJing

Transformation of 07/31/13 works. We will do a PCR Colony.

We took a single colony and resuspend in 10µL H2O. For each Biobrick, we did 25 PCR Colonies.

Used quantities :

PCR preparation mix for 25 different colonies including:

    • Oligo 44 : 3.5µL
    • Oligo 45 : 3.5µL
    • Buffer Dream Taq : 70µL
    • dNTP : 28µL
    • Dream Taq : 5µL
    • H2O : 590µL

PCR reaction:

  • DNA : 2µL of resuspend colony
  • Mix PCR  :23µL

Total volume: 25µL

PCR Program :

PsPCR0508.jpg


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