Team:Paris Saclay/Notebook/August/14

From 2013.igem.org

(Difference between revisions)
(→2 - Extraction of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006 from DH5α)
(→9 - Gel purification of the digestion of BBa_K1155003 and BBa_K1155007 by XBaI/PstI)
 
(44 intermediate revisions not shown)
Line 7: Line 7:
==='''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 - Gel purification of the digestion of Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI'''====  
+
===='''1 - Gel purification of the digestion of BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI'''====  
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 :  
* NarG : 10.5ng/µL
* NarG : 10.5ng/µL
* NarK : 16.8ng/µL
* NarK : 16.8ng/µL
-
* NirB : 24.3
+
* NirB : 24.3ng/µl
{|
{|
Line 25: Line 25:
|}
|}
-
===='''2 - Extraction of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006 from DH5α'''====  
+
===='''2 - Extraction of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 from DH5α'''====  
XiaoJing
XiaoJing
-
Protocol : [[Team:Paris_Saclay/extraction|Hight copy plamid extraction]]
+
Protocol : [[Team:Paris_Saclay/extraction|High-copy plamid extraction]]
Nanodrop :  
Nanodrop :  
Line 35: Line 35:
* NarG : 80.7ng/µL
* NarG : 80.7ng/µL
* Nir B :65.8ng/µL
* Nir B :65.8ng/µL
-
* Pfnr : 227ng/µL
+
* Pndh* : 227ng/µL
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
The extraction was good. We will digest  Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006.
+
The extraction was good. We will digest  BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006.
|}
|}
-
====3 - Digestion of Bba_K1155004,Bba_K1155005, Bba_K1155006 by EcoRI/SpeI====
+
====3 - Digestion of BBa_K1155004,BBa_K1155005, BBa_K1155006 by EcoRI/SpeI====
Nadia
Nadia
Line 54: Line 54:
We let the digestion at 37°C during 15 minutes.
We let the digestion at 37°C during 15 minutes.
-
====4 - Electrophoresis to check the digestion of Bba_K1155004,Bba_K1155005, Bba_K1155006 by EcoRI/SpeI====
+
====4 - Electrophoresis to check the digestion of BBa_K1155004, BBa_K1155005, BBa_K1155006 by EcoRI/SpeI====
Anaïs, Nadia
Anaïs, Nadia
Line 62: Line 62:
| 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_K1155006 digested by EcoRI/SpeI+1µl of 6X loading dye
+
*Well 2 : 5µL of BBa_K1155006 digested by EcoRI/SpeI +1µl of 6X loading dye
-
*Well 3 : 5µL of BBa_K1155005 digested by EcoRI/SpeI+1µl of 6X loading dye
+
*Well 3 : 5µL of BBa_K1155005 digested by EcoRI/SpeI +1µl of 6X loading dye
-
*Well 4 : 5µL of BBa_K1155004 digested by EcoRI/SpeI+1µl of 6X loading dye
+
*Well 4 : 5µL of BBa_K1155004 digested by EcoRI/SpeI +1µl of 6X loading dye
*Gel : 1%
*Gel : 1%
|}
|}
Line 70: Line 70:
Expected sizes :
Expected sizes :
-
*PSB1C3 : 2070kb
+
*pSB1C3 : 2070kb
*NarK, NarG, NirB : 200kb
*NarK, NarG, NirB : 200kb
Line 78: Line 78:
|}
|}
-
====5 - Digestion of Bba_K1155000, Bba_K1155004,Bba_K1155005, Bba_K1155006 by SpeI and EcoRI/SpeI====
+
====5 - Digestion of BBa_K1155000, BBa_K1155004,BBa_K1155005, BBa_K1155006 by SpeI and EcoRI/SpeI====
Anaïs, Nadia
Anaïs, Nadia
Line 94: Line 94:
**H20 : 3µL  
**H20 : 3µL  
-
We let digestions at 37°C during 10 minutes ??????
+
We let digestions at 37°C during 10 minutes.
-
====6 - Electrophoresis to check the digestion of Bba_K1155000, Bba_K1155004,Bba_K1155005, Bba_K1155006 by SpeI and EcoRI/SpeI====
+
====6 - Electrophoresis to check the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI and EcoRI/SpeI====
Anaïs, Nadia
Anaïs, Nadia
Line 102: Line 102:
| style="width:350px;border:1px solid black;" | [[File:PSgel3_1408.jpg]]
| style="width:350px;border:1px solid black;" | [[File:PSgel3_1408.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
*Well 1 : 5µL of BBa_K1155000 digested by SpeI+1µl of 6X loading dye
+
*Well 1 : 5µL of BBa_K1155000 digested by SpeI + 1µl of 6X loading dye
-
*Well 2 : 5µL of BBa_K1155006 digested by SpeI+1µl of 6X loading dye
+
*Well 2 : 5µL of BBa_K1155006 digested by SpeI + 1µl of 6X loading dye
-
*Well 3 : 5µL of BBa_K1155005 digested by SpeI+1µl of 6X loading dye
+
*Well 3 : 5µL of BBa_K1155005 digested by SpeI + 1µl of 6X loading dye
-
*Well 4 : 5µL of BBa_K1155004 digested by SpeI+1µl of 6X loading dye
+
*Well 4 : 5µL of BBa_K1155004 digested by SpeI + 1µl of 6X loading dye
-
*Well 5 : 6µL DNA Ladder
+
*Well 5 : 6µL DNA Ladder  
-
*Well 6 : 5µL of BBa_K1155000 digested by EcoRI/SpeI+1µl of 6X loading dye
+
*Well 6 : 5µL of BBa_K1155000 digested by EcoRI/Spe I + 1µl of 6X loading dye
-
*Well 7 : 5µL of BBa_K1155006 digested by EcoRI/SpeI+1µl of 6X loading dye
+
*Well 7 : 5µL of BBa_K1155006 digested by EcoRI/Spe I + 1µl of 6X loading dye
-
*Well 8 : 5µL of BBa_K1155005 digested by EcoRI/SpeI+1µl of 6X loading dye
+
*Well 8 : 5µL of BBa_K1155005 digested by EcoRI/Spe I + 1µl of 6X loading dye
-
*Well 9 : 5µL of BBa_K1155004 digested by EcoRI/SpeI+1µl of 6X loading dye
+
*Well 9 : 5µL of BBa_K1155004 digested by EcoRI/Spe I + 1µl of 6X loading dye
*Gel : 1%
*Gel : 1%
|}
|}
Line 116: Line 116:
Expected sizes :
Expected sizes :
-
*Pfnr in PSB1C3 : ...
+
*Pndh* in pSB1C3 : 2200bp
-
*Nar K in PSB1C3, NarG in PSB1C3, NirB in PSB1C3 : ...
+
*Nar K in pSB1C3, NarG in pSB1C3, NirB in pSB1C3 : 2270bp
-
*PSB1C3 : 2070kb
+
*pSB1C3 : 2070kb
*NarK, NarG, NirB : 200kb
*NarK, NarG, NirB : 200kb
-
*Pfnr : ...
+
*Pndh* : 111bp
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
Well 1 : we have done one digestion so we have to obtain one fragment, we have two stripes, the digestion of Bba_K1155000 by SpeI wasn't good.
+
Well 1 : we have done one digestion so we have to obtain one fragment, we have two stripes, the digestion of BBa_K1155000 by SpeI wasn't good.
-
Well 2, 3, 4, 6, 7 : we obtain NarG, NarK and NirB in PSB1C3 and Pfnr, NarK fragments at the right size, we can purify it.
+
Well 2, 3, 4, 6, 7 : we obtain NarG, NarK and NirB in pSB1C3 and Pndh*, NarK fragments at the right size, we can purify it.
-
Well 8, 9 : we have done two digestions so we have to obtain two fragments, we have only one stripe, the digestion of Bba_K1155005 and Bba_K1155004 by EcoRI/SpeI wasn't good.
+
Well 8, 9 : we have done two digestions so we have to obtain two fragments, we have only one stripe, the digestion of BBa_K1155005 and BBa_K1155004 by EcoRI/SpeI wasn't good.
|}
|}
-
====7 - Gel purification of the digestion of Bba_K1155000 and Bba_K1155006 by EcoRI/SpeI and Bba_K1155004, Bba_K1155005 and Bba_K1155006 by SpeI====
+
====7 - Gel purification of the digestion of BBa_K1155000 and BBa_K1155006 by EcoRI/SpeI and BBa_K1155004, BBa_K1155005 and BBa_K1155006 by SpeI====
Anaïs, Nadia
Anaïs, Nadia
-
Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]]
+
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
{|
{|
-
| style="width:350px;border:1px solid black;" | [[]]
+
| style="width:350px;border:1px solid black;" | [[File:Psgel31408.jpg| 350px]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
*The cutting for the gel purification was good.
*The cutting for the gel purification was good.
Line 142: Line 142:
Nanodrop :
Nanodrop :
-
* Pfnr : 13.7ng/µL
+
* Pndh* : 13.7ng/µL
* NarK : 27.9ng/µL
* NarK : 27.9ng/µL
* NarK in PSB1C3 : 87.1ng/µL
* NarK in PSB1C3 : 87.1ng/µL
Line 150: Line 150:
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
CONCLUSION pour Pfnr faible quantité d'où on recommence plus tard ?,?????
+
The purification was good. We will ligate them.
|}
|}
-
====8 - Gel purification of the digestion of Bba_K1155003 and Bba_K1155007 by XBaI/PstI====
+
====8 - Electrophoresis of the digestion of BBa_K1155003, BBa_K1155007 by XBaI/PstI====
 +
 
Nadia
Nadia
{|
{|
-
| style="width:350px;border:1px solid black;" | [[]]
+
| style="width:350px;border:1px solid black;" | [[File:Psgel51408.jpg|350px]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
*Thanks to electrophoresis we separated all the quantity of our digestion. 
+
* Well 1 : 6µL of DNA Ladder
 +
* Well 2 : 20µL of BBa_K1155003 digested by XBaI/PstI + 4µL of 6X loading dye
 +
* Well 3 : 20µL of BBa_K1155003 digested by XBaI/PstI + 4µL of 6X loading dye
 +
* Well 4 : 20µL of BBa_K1155007 digested by XBaI/PstI + 4µL of 6X loading dye
 +
* Well 5 : 20µL of BBa_K1155007 digested by XBaI/PstI + 4µL of 6X loading dye
|}
|}
 +
 +
Expected sizes :
 +
* RBS_LacZ-Term : 3500bp
 +
* RBS_AmilCP-Term : 824bp
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtained fragments at the right size. We will purify it.
 +
|}
 +
 +
====9 - Gel purification of the digestion of BBa_K1155003 and BBa_K1155007 by XBaI/PstI====
 +
Nadia
Nanodrop :  
Nanodrop :  
Line 168: Line 185:
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
WE obtain few quantities of plasmid that's why we will make an Ethanol precipitation to concentrated it.
+
We obtained low quantities of plasmid that's why we will make an Ethanol precipitation to concentrate it.
|}
|}
 +
 +
====10 - Glycerol stock of MG1655Z1 Δfnr::Km====
 +
 +
XioaJing
 +
 +
Used quantities :
 +
* Glycerol : 500 µL
 +
* MG1655Z1 Δfnr::Km: 1 mL
 +
 +
We stored the bacteria at -20°C.
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
Line 180: Line 207:
* RBS-BphR2 :  
* RBS-BphR2 :  
-
** PSB1C3 : 3µL
+
** pSB1C3 : 3µL
** BphR2 Part I : 1µL  
** BphR2 Part I : 1µL  
** BphR2 Part II : 1µL  
** BphR2 Part II : 1µL  
-
** Gibson mix : 15µL ???????????????
+
** Gibson mix : 15µL  
* FNR :  
* FNR :  
-
** PSB1C3 : 3µL
+
** pSB1C3 : 3µL
** FNR Part I : 1µL
** FNR Part I : 1µL
** FNR Part II : 1µL
** FNR Part II : 1µL
-
** Gisbon mix : 15µL ???????????????
+
** Gisbon mix : 15µL  
* RBS-FNR :
* RBS-FNR :
-
** PSB1C3 : 3µL
+
** pSB1C3 : 3µL
** RBS-FNR Part I : 1µL
** RBS-FNR Part I : 1µL
** FNR Part II : 1µL
** FNR Part II : 1µL
-
** Gibson mix : 15µL ????????????
+
** Gibson mix : 15µL  
-
We let these mix at 50°C during 1h. Then we the these mix at 4°C during the week end.
+
We incubate these mix at 50°C during 1h inside PCR machine. Then we keep these mix at 4°C during the week end.
===='''2 - Electrophoresis of PCR products : BphR2 Part I'''====  
===='''2 - Electrophoresis of PCR products : BphR2 Part I'''====  
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{|
{|
-
| style="width:350px;border:1px solid black;" | [[]]
+
| style="width:350px;border:1px solid black;" | [[File:Psgel41408.jpg|350px]]
| 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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{|
{|
-
| style="width:350px;border:1px solid black;" | [[]]
+
| style="width:350px;border:1px solid black;" | [[File:Psgel61408.jpg|250px]]
| 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 BphR2 Part I+1µL of 6X loading dye
+
*Well 2 : 5µL of BphR2 Part I + 1µL of 6X loading dye
-
*Well 3 : 5µL of BphR2 Part I+1µL of 6X loading dye
+
*Well 3 : 5µL of BphR2 Part I + 1µL of 6X loading dye
|}
|}
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* dNTP : 1µL
* dNTP : 1µL
* Phusion : 1µL
* Phusion : 1µL
-
* DMS9 : 2µL ??????????????????????????????????
+
* DMS0 : 2µL  
* H2O : 31µL   
* H2O : 31µL   
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[[File:Pstest.jpg|400px]]
[[File:Pstest.jpg|400px]]
 +
 +
 +
{| border="1" align="center"
 +
|[[Team:Paris Saclay/Notebook/August/13|<big>Previous day</big>]]
 +
 +
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 +
 +
|[[Team:Paris Saclay/Notebook/August/19|<big>Next day</big>]]
 +
|}
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 01:29, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 14

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Gel purification of the digestion of BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI

XiaoJing

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

Nanodrop :

  • NarG : 10.5ng/µL
  • NarK : 16.8ng/µL
  • NirB : 24.3ng/µl

We lost our plasmids. We will do the digestion again.

2 - Extraction of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 from DH5α

XiaoJing

Protocol : High-copy plamid extraction

Nanodrop :

  • NarK: 89.8ng/µL
  • NarG : 80.7ng/µL
  • Nir B :65.8ng/µL
  • Pndh* : 227ng/µL

The extraction was good. We will digest BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006.

3 - Digestion of BBa_K1155004,BBa_K1155005, BBa_K1155006 by EcoRI/SpeI

Nadia

  • Buffer FD : 2µL
  • H2O : 6µL
  • DNA : 10µL
  • SpeI FD : 1µL
  • EcoRI FD : 1µL

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

4 - Electrophoresis to check the digestion of BBa_K1155004, BBa_K1155005, BBa_K1155006 by EcoRI/SpeI

Anaïs, Nadia

PSgel2 1408.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BBa_K1155006 digested by EcoRI/SpeI +1µl of 6X loading dye
  • Well 3 : 5µL of BBa_K1155005 digested by EcoRI/SpeI +1µl of 6X loading dye
  • Well 4 : 5µL of BBa_K1155004 digested by EcoRI/SpeI +1µl of 6X loading dye
  • Gel : 1%

Expected sizes :

  • pSB1C3 : 2070kb
  • NarK, NarG, NirB : 200kb

We obtained NarK, NarG, NirB fragments at the right size but in very few quantity. We do it again but this time we will use more quantity of enzymes.

5 - Digestion of BBa_K1155000, BBa_K1155004,BBa_K1155005, BBa_K1155006 by SpeI and EcoRI/SpeI

Anaïs, Nadia

  • Digestion by SpeI :
    • Buffer : 2µL
    • SpeI : 2µL
    • ADN : 15µL
    • H20 : 1µL
  • Digestion by EcoRI and SpeI :
    • Buffer : 3µL
    • SpeI : 2µL
    • EcoRI : 2µL
    • ADN : 20µL
    • H20 : 3µL

We let digestions at 37°C during 10 minutes.

6 - Electrophoresis to check the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI and EcoRI/SpeI

Anaïs, Nadia

PSgel3 1408.jpg
  • Well 1 : 5µL of BBa_K1155000 digested by SpeI + 1µl of 6X loading dye
  • Well 2 : 5µL of BBa_K1155006 digested by SpeI + 1µl of 6X loading dye
  • Well 3 : 5µL of BBa_K1155005 digested by SpeI + 1µl of 6X loading dye
  • Well 4 : 5µL of BBa_K1155004 digested by SpeI + 1µl of 6X loading dye
  • Well 5 : 6µL DNA Ladder
  • Well 6 : 5µL of BBa_K1155000 digested by EcoRI/Spe I + 1µl of 6X loading dye
  • Well 7 : 5µL of BBa_K1155006 digested by EcoRI/Spe I + 1µl of 6X loading dye
  • Well 8 : 5µL of BBa_K1155005 digested by EcoRI/Spe I + 1µl of 6X loading dye
  • Well 9 : 5µL of BBa_K1155004 digested by EcoRI/Spe I + 1µl of 6X loading dye
  • Gel : 1%

Expected sizes :

  • Pndh* in pSB1C3 : 2200bp
  • Nar K in pSB1C3, NarG in pSB1C3, NirB in pSB1C3 : 2270bp
  • pSB1C3 : 2070kb
  • NarK, NarG, NirB : 200kb
  • Pndh* : 111bp

Well 1 : we have done one digestion so we have to obtain one fragment, we have two stripes, the digestion of BBa_K1155000 by SpeI wasn't good. Well 2, 3, 4, 6, 7 : we obtain NarG, NarK and NirB in pSB1C3 and Pndh*, NarK fragments at the right size, we can purify it. Well 8, 9 : we have done two digestions so we have to obtain two fragments, we have only one stripe, the digestion of BBa_K1155005 and BBa_K1155004 by EcoRI/SpeI wasn't good.

7 - Gel purification of the digestion of BBa_K1155000 and BBa_K1155006 by EcoRI/SpeI and BBa_K1155004, BBa_K1155005 and BBa_K1155006 by SpeI

Anaïs, Nadia

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

Psgel31408.jpg
  • The cutting for the gel purification was good.

Nanodrop :

  • Pndh* : 13.7ng/µL
  • NarK : 27.9ng/µL
  • NarK in PSB1C3 : 87.1ng/µL
  • NarG in PSB1C3 : 39.2ng/µL
  • NirB in PSB1C3 : 37.7ng/µL

The purification was good. We will ligate them.

8 - Electrophoresis of the digestion of BBa_K1155003, BBa_K1155007 by XBaI/PstI

Nadia

Psgel51408.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 : 20µL of BBa_K1155003 digested by XBaI/PstI + 4µL of 6X loading dye
  • Well 3 : 20µL of BBa_K1155003 digested by XBaI/PstI + 4µL of 6X loading dye
  • Well 4 : 20µL of BBa_K1155007 digested by XBaI/PstI + 4µL of 6X loading dye
  • Well 5 : 20µL of BBa_K1155007 digested by XBaI/PstI + 4µL of 6X loading dye

Expected sizes :

  • RBS_LacZ-Term : 3500bp
  • RBS_AmilCP-Term : 824bp

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

9 - Gel purification of the digestion of BBa_K1155003 and BBa_K1155007 by XBaI/PstI

Nadia

Nanodrop :

  • RSB-LacZ-Term : 59.6ng/µL
  • RBS-AmilCP-Term : 28.7ng/µL

We obtained low quantities of plasmid that's why we will make an Ethanol precipitation to concentrate it.

10 - Glycerol stock of MG1655Z1 Δfnr::Km

XioaJing

Used quantities :

  • Glycerol : 500 µL
  • MG1655Z1 Δfnr::Km: 1 mL

We stored the bacteria at -20°C.

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

Objective : obtaining FNR and BphR2 proteins

1 - Gibson assembly

Used quantities :

  • RBS-BphR2 :
    • pSB1C3 : 3µL
    • BphR2 Part I : 1µL
    • BphR2 Part II : 1µL
    • Gibson mix : 15µL
  • FNR :
    • pSB1C3 : 3µL
    • FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gisbon mix : 15µL
  • RBS-FNR :
    • pSB1C3 : 3µL
    • RBS-FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gibson mix : 15µL

We incubate these mix at 50°C during 1h inside PCR machine. Then we keep these mix at 4°C during the week end.

2 - Electrophoresis of PCR products : BphR2 Part I

Damir

Psgel41408.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BphR2 Part I+1µL of 6X loading dye
Psgel61408.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BphR2 Part I + 1µL of 6X loading dye
  • Well 3 : 5µL of BphR2 Part I + 1µL of 6X loading dye

Expected sizes :

  • BphR2 Part I : 178kb

On the first gel, all deposits disappear so we did the electrophoresis again. On the second gel, we didn't obtain stripes at the good size. We do the PCR again using new quantities and a new PCR program.

3 - 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


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