Team:Paris Saclay/Notebook/August/13

From 2013.igem.org

(Difference between revisions)
Line 35: Line 35:
* Bba_K1155000 :  
* Bba_K1155000 :  
-
** Buffer : 5µL
+
** Buffer FD : 5µL
** H2O : 39µL
** H2O : 39µL
** DNA : 5µL
** DNA : 5µL
-
** SpeI : 1µL
+
** SpeI FD : 1µL
* Bba_K1155000 :  
* Bba_K1155000 :  
-
** Buffer : 5µL
+
** Buffer FD : 5µL
** H2O : 38µL
** H2O : 38µL
** DNA : 5µL
** DNA : 5µL
-
** SpeI : 1µL
+
** SpeI FD : 1µL
-
**EcoRI : 1µL
+
**EcoRI FD : 1µL
* Bba_K1155004, Bba_K1155005, bba_K1155006 :  
* Bba_K1155004, Bba_K1155005, bba_K1155006 :  
-
** Buffer : 2µL
+
** Buffer FD : 2µL
** H2O : 7µL
** H2O : 7µL
** DNA : 10µL
** DNA : 10µL
-
** SpeI : 1µL
+
** SpeI FD : 1µL
* Bba_K1155004, Bba_K1155005, bba_K1155006 :  
* Bba_K1155004, Bba_K1155005, bba_K1155006 :  
-
** Buffer : 2µL
+
** Buffer FD : 2µL
** H2O : 6µL
** H2O : 6µL
** DNA : 10µL
** DNA : 10µL
-
** SpeI : 1µL
+
** SpeI FD : 1µL
-
** EcoRI : 1µL
+
** EcoRI FD : 1µL
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 +
=====4 - Digestion of Bba_J04450 by EcoRI and PstI  =====
 +
Anaïs
 +
* Buffer FD: 2µL
 +
* H2O : 6µL
 +
* DNA : 10µL
 +
* EcoRI FD : 1µL
 +
* PstI FD : 1µL
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===

Revision as of 11:10, 21 August 2013

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Contents

Notebook : August 13

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining ...

1 - Electrophoresis to check the digestion of Bba_K1155000 by SpeI and PstI

Nadia

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL Bba_K1155000 digested by SpeI and PstI+1µl of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • Pfnr : ...
  • PSB1C3 : ...

We can't see any band for Bba_K1155000 digestion. The digestion failed because we used the wrong enzymes. We will do it again with the good ones.

2 - Digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI and by SpeI/EcoRI

Anaïs, Nadia

  • Bba_K1155000 :
    • Buffer FD : 5µL
    • H2O : 39µL
    • DNA : 5µL
    • SpeI FD : 1µL
  • Bba_K1155000 :
    • Buffer FD : 5µL
    • H2O : 38µL
    • DNA : 5µL
    • SpeI FD : 1µL
    • EcoRI FD : 1µL
  • Bba_K1155004, Bba_K1155005, bba_K1155006 :
    • Buffer FD : 2µL
    • H2O : 7µL
    • DNA : 10µL
    • SpeI FD : 1µL
  • Bba_K1155004, Bba_K1155005, bba_K1155006 :
    • Buffer FD : 2µL
    • H2O : 6µL
    • DNA : 10µL
    • SpeI FD : 1µL
    • EcoRI FD : 1µL


3 - Electrophoresis to check the digestion of Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI and by SpeI/EcoRI

Anaïs, Nadia

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL Bba_K1155006 digested by SpeI+1µl of 6X loading dye
  • Well 3 : 5µL Bba_K1155005 digested by SpeI+1µl of 6X loading dye
  • Well 4 : 5µL Bba_K1155004 digested by SpeI+1µl of 6X loading dye
  • Well 5 : 5µL Bba_K1155006 digested by EcoRI/SpeI+1µl of 6X loading dye
  • Well 6 : 5µL Bba_K1155005 digested by EcoRI/SpeI+1µl of 6X loading dye
  • Well 7 : 5µL Bba_K1155004 digested by EcoRI/SpeI+1µl of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • NarK, Nar G, NirB : 200kb
  • PSB1C3 : ...
  • NarK in PSB1C3, NarG in PSB1C3, NirB in PSB1C3 : ...

We lost all our digestion product so we will do it again ??????????????????

4 - Digestion of Bba_J04450 by EcoRI and PstI

Anaïs

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

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

Objective : obtaining FNR and BphR2 proteins

1 - PCR of FRN Part I, FNR Part II, BphR2 Part I

Anaïs

Protocol : 07/30/13

2 - Electrophoresis of PCR products : FRN Part I, FNR Part II, BphR2 Part I

Damir

[[]]
  • Well 1 : 5µL FNR Part II+1µl of 6X loading dye
  • Well 2 : 5µL BphR2 Part I+1µl of 6X loading dye
  • Well 3 : 5µL FNR Part I+1µl of 6X loading dye
  • Well 4 : 6µL DNA Ladder
  • Gel : 1%
[[]]
  • Well 1 : 5µL FNR Part II+1µl of 6X loading dye
  • Well 2 : 5µL BphR2 Part I+1µl of 6X loading dye
  • Well 3 : 5µL FNR Part I+1µl of 6X loading dye
  • Well 4 : 6µL DNA Ladder
  • Gel : 1%

Expected sizes :

  • FNR part I : 597 kb
  • FNR part II : 200 kb
  • BphR2 part I : 178 kb

It's impossible to read the first gel. We do it again. In the second gel, we obtain FNR Part I and FNR Part II fragments at the right size. We can purify it. We also obtain BphR2 frangment at the right size but it was mix with other DNA frangments. We will try to make a gel purification of it.

3 - Gel purification of PCR product : BphR2 Part I

Damir


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