Team:Paris Saclay/Notebook/August/22

From 2013.igem.org

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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
+
===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
-
===='''1 - Plasmid extraction of Bba_K1155000 from DH5α'''====
+
===='''1 - Plasmid extraction of BBa_K1155000 from DH5α'''====
Nguyen
Nguyen
-
Protocol : [[Team:Paris_Saclay/Protocols/plasmid extraction|Plasmid extraction]]
+
Protocol : [[Team:Paris_Saclay/extraction|High-copy plamid extraction]]
Nanodrop :  
Nanodrop :  
-
* Bba_K1155000 : 175ng/µL
+
* BBa_K1155000 : 175ng/µL
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
CONCLUSION !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
+
The extraction was good. We will digested the plasmid.
|}
|}
-
===='''2 - Digestion of Bba_K1155000 by SpeI'''====
+
===='''2 - Digestion of BBa_K1155000 by SpeI'''====
 +
 
-
Nguyen
 
Used quantities :  
Used quantities :  
-
* Bba_K1155000 : 10µL
+
* BBa_K1155000 : 10µL
* Buffer FD : 2µL
* Buffer FD : 2µL
-
* Spe I : 2µL
+
* SpeI : 2µL
* H2O : 6 µL
* H2O : 6 µL
-
We let digestions at 37°C during 10 minutes ??????
+
We let digestions at 37°C during 10 minutes.
-
 
+
-
QU'EN A T'ON FAIT PAR LA SUITE ?????????
+
-
((((((((((((((((((===='''Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3'''====
+
===='''Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in pSB3K3'''====
-
===='''1 - Gel purification of the digestion of Bba_J04450 by EcoRI/PstI '''====
+
===='''1 - Gel purification of the digestion of BBa_J04450 by EcoRI/PstI '''====
XiaoJing
XiaoJing
-
Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]]
+
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
Nanodrop :  
Nanodrop :  
-
* PSB3K3 : 4ng/µL
+
* pSB3K3 : 4ng/µL
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
The Nanodrop gives us a very few quantity of PSB3K3 so we decided to check it with a first electrophoresis.  
+
The Nanodrop gives us a very few quantity of pSB3K3 so we decided to check it with a first electrophoresis.  
|}
|}
 +
 +
===='''2 - Electrophoresis of gel purification of the digestion of BBa_J04450 by EcoRI/PstI '''====
 +
 +
XiaoJing
{|
{|
-
| style="width:350px;border:1px solid black;" |]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel12208.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
* Well 1 : 6µL DNA Ladder
* Well 1 : 6µL DNA Ladder
-
* Well 2 : 5µL of Bba_J04450 digested by EcoRI/PstI+1µl of 6X loading dye
+
* Well 2 : 5µL of BBa_J04450 digested by EcoRI/Pst I + 1µl of 6X loading dye
* Gel : 1%
* Gel : 1%
|}
|}
Expect sizes :  
Expect sizes :  
-
* PSB3K3 : 2750 bp
+
* pSB3K3 : 2750 bp
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
We can see anything with the fisrt electrophoresis that's why we made an EtOH precipitation.
+
We can't see anything with the fisrt electrophoresis that's why we made an EtOH precipitation.
|}
|}
-
Protocol : [[Team:Paris_Saclay/Protocols/EtOH precipitation|EtOH precipitation]]
+
===='''3 - Ethanol precipitation of the digestion of BBa_J04450 by EcoRI/PstI '''====
-
We used 34µL of DNA. ))))))))))))))))))))))
+
Protocol : [[Team:Paris_Saclay/ethanol|EtOH precipitation]]
 +
 
 +
We used 34µL of DNA.
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Used quantities :  
Used quantities :  
-
*
+
* Oligo 54F : 2µL
 +
* Oligo 55R : 2µL
 +
* DNA : 1µL
 +
* Buffer Phusion : 10µL
 +
* dNTP : 1µL
 +
* Phusion : 1µL
 +
* DMS0 : 2µL
 +
* H2O : 31µL 
-
PCR Program :  
+
PCR program :
 +
 
 +
[[File:Pstest.jpg|400px]]
===='''2 - Electrophoresis of PCR product : BphR2 Part I'''====
===='''2 - Electrophoresis of PCR product : BphR2 Part I'''====
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{|
{|
-
| style="width:350px;border:1px solid black;" |]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel22208.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
* Well 1 : 6µL DNA Ladder
* Well 1 : 6µL DNA Ladder
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Expected sizes :  
Expected sizes :  
-
Bphr2 Part I : 178 kb  
+
* BphR2 Part I : 178 kb  
{|
{|
| 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 can purify it.
+
We obtained fragments at the right size. We can purify it.
|}
|}
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Damir
Damir
-
Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]]
+
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
Nanodrop :  
Nanodrop :  
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* RBS-BphR2 Part I, tube 2 : 75ng/µL
* RBS-BphR2 Part I, tube 2 : 75ng/µL
-
{|
+
 
-
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
+
{| border="1" align="center"
-
CONCLUSION !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
+
|[[Team:Paris Saclay/Notebook/August/21|<big>Previous day</big>]]
 +
 
 +
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 +
 
 +
|[[Team:Paris Saclay/Notebook/August/23|<big>Next day</big>]]
|}
|}
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 01:16, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 22

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Plasmid extraction of BBa_K1155000 from DH5α

Nguyen

Protocol : High-copy plamid extraction

Nanodrop :

  • BBa_K1155000 : 175ng/µL

The extraction was good. We will digested the plasmid.

2 - Digestion of BBa_K1155000 by SpeI

Used quantities :

  • BBa_K1155000 : 10µL
  • Buffer FD : 2µL
  • SpeI : 2µL
  • H2O : 6 µL

We let digestions at 37°C during 10 minutes.

Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in pSB3K3

1 - Gel purification of the digestion of BBa_J04450 by EcoRI/PstI

XiaoJing

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

Nanodrop :

  • pSB3K3 : 4ng/µL

The Nanodrop gives us a very few quantity of pSB3K3 so we decided to check it with a first electrophoresis.

2 - Electrophoresis of gel purification of the digestion of BBa_J04450 by EcoRI/PstI

XiaoJing

Psgel12208.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BBa_J04450 digested by EcoRI/Pst I + 1µl of 6X loading dye
  • Gel : 1%

Expect sizes :

  • pSB3K3 : 2750 bp

We can't see anything with the fisrt electrophoresis that's why we made an EtOH precipitation.

3 - Ethanol precipitation of the digestion of BBa_J04450 by EcoRI/PstI

Protocol : EtOH precipitation

We used 34µL of DNA.


A - Aerobic/Anaerobic regulation system / B - PCB sensor system

Objective : obtaining BphR2 protein

1 - PCR of BphR2 Part I

Damir

Used quantities :

  • Oligo 54F : 2µL
  • Oligo 55R : 2µL
  • DNA : 1µL
  • Buffer Phusion : 10µL
  • dNTP : 1µL
  • Phusion : 1µL
  • DMS0 : 2µL
  • H2O : 31µL

PCR program :

Pstest.jpg

2 - Electrophoresis of PCR product : BphR2 Part I

Damir

Psgel22208.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 40µL of BphR2 Part I+8µl of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • BphR2 Part I : 178 kb

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

3 - Gel purification of PCR product : BphR2 Part I

Damir

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

Nanodrop :

  • RBS-BphR2 Part I, tube 1 : 42ng/µL
  • RBS-BphR2 Part I, tube 2 : 75ng/µL


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