Latest revision as of 19:26, 3 October 2013

Navigation Home Team Project Labwork Notebook Human practices

1 Notebook : August 12
- 1.1 Lab work
  - 1.1.1 A - Aerobic/Anaerobic regulation system
  - 1.1.2 A - Aerobic/Anaerobic regulation system / B - PCB sensor system
    - 1.1.2.1 Objective : obtaining FNR and BphR2 proteins
    - 1.1.2.2 1 - Electrophoresis of PCR products : RBS-BphR2 Part I, BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I and FNR Part II

Notebook : August 12

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000

1 - Digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBaI/PstI

Anaïs, Nadia, XiaoJing

Used quantities :

BBa_K1155000 :
- Buffer FD : 5µL
- H2O : 38µL
- DNA : 5µL
- SpeI FD : 1µL
- PstI FD : 1µL

BBa_K1155007 :
- Buffer FD : 5µL
- H2O : 23µL
- DNA : 20µL
- XBal FD : 1µL
- PstI FD : 1µL

BBa_K1155003 :
- Buffer FD : 5µL
- H2O : 33µL
- DNA : 10µL
- XBal FD : 1µL
- PstI FD : 1µL

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

2 - Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBalI/PstI

Nadia

Well 1 : 6µL DNA Ladder
Well 2 : 5µL BBa_K1155000 digested by SpeI/PstI + 1µl of 6X loading dye
Well 3 : 5µL BBa_K1155007 digested by XBaI/PstI + 1µl of 6X loading dye
Well 4 : 5µL BBa_K1155003 digested by xBaI/PstI + 1µl of 6X loading dye
Gel : 0.8%

Expected sizes :

Pndh* : 111bp
RBS_LacZ_Term : 3500 kb
RBS_AmilCP_Term : 824 bp
pSB1C3 : 2070bp

We can't see any band for BBa_K1155000 digestion. The digestion failed. We will do it again. We obtain RBS-LacZ-Term and RBS-AmilCP-Term fragments. The digestion was good. We will purify it.

3 - Digestion of BBa_K1155000 by Spe I/Pst I

Anaïs, Nadia

Used quantities :

Buffer FD : 5µL
H2O : 38µL
DNA : 5µL
SpeI FD : 1µL
PstI FD : 1µL

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

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

Objective : obtaining FNR and BphR2 proteins

1 - Electrophoresis of PCR products : RBS-BphR2 Part I, BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I and FNR Part II

Damir

Well 1 : 6µL DNA Ladder
Well 2 : 5µL RBS-BphR2 Part I +1µl of 6X loading dye
Well 3 : 5µL BphR2 Part II +1µl of 6X loading dye
Well 4 : 5µL FNR Part I +1µl of 6X loading dye
Well 5 : 5µL FNR Part II +1µl of 6X loading dye
Well 6 : 5µL RBS-FNR Part I +1µl of 6X loading dye
Well 7 : 5µL BphR2 Part I +1µl of 6X loading dye
Gel : 0.8%

Expected size

RBS-BphR2 Part I : 197 kb
BphR2 Part II : 790 kb
FNR Part I : 597 kb
FNR Part II : 200 kb
RBS-FNR Part I : 615 kb
BphR2 Part I : 178 kb

We can't see FNR Part I, FNR Part II and BphR2 Part I fragments at the good size. We will make the PCR again. We obtain RBS-BphR2 Part I, BphR2 Part II, RBS-FNR Part I frangments at the right size thanks to the PCR. We will purify it.

Previous day

Back to calendar

Next day

@@ Line 7: / Line 7: @@
 ==='''A - Aerobic/Anaerobic regulation system'''===
-'''Obtaining Pfnr_RBS-LacZ-Term in PSB1C3'''
+===='''Objective : characterize BBa_K1155000'''====
-===='''1 - Digestion of Bba_K1155000, Bba_K1155007 and Bba_K1155003'''====
+===='''1 - Digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBaI/PstI'''====
 Anaïs, Nadia, XiaoJing
-Protocol : [[Team:Paris_Saclay/Protocols/digestion|digestion]]
+Used quantities :
-Used quantities :
+* BBa_K1155000 :
-* Pfnr in PSB1C3 (Bba_K1155000) :
+** Buffer FD : 5µL
-** Buffer : 5µL
 ** H2O : 38µL
-** Pfnr : 5µL
+** DNA : 5µL
-** SpeI : 1µL
+** SpeI FD : 1µL
-** PstI : 1µL
+** PstI FD : 1µL
-* RBS-LacZ-Term in PSB1C3 (Bba_K1155007) :
+* BBa_K1155007 :
-** Buffer : 5µL
+** Buffer FD : 5µL
 ** H2O : 23µL
-** RBS-LacZ-Term : 20µL
+** DNA : 20µL
-** XBal : 1µL
+** XBal FD : 1µL
-** PstI : 1µL
+** PstI FD : 1µL
-* RBS-AmilCP-Term in PSB1C3 (Bba_K1155003) :
+* BBa_K1155003 :
-** Buffer : 5µL
+** Buffer FD : 5µL
 ** H2O : 33µL
-** RBS-AmilCP-Term : 10µL
+** DNA : 10µL
-** XBal : 1µL
+** XBal FD : 1µL
-** PstI : 1µL
+** PstI FD : 1µL
-===='''2 - Electrophoresis of digestion pieces'''====
+We incubated the digestion at 37°C during 15 minutes.
+===='''2 - Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBalI/PstI'''====
+Nadia
-We gonna check if the digestion was good by control the size of digestion pieces.
 {|
-| style="width:350px;border:1px solid black;" |[[File:Psdigestion12.jpg|350px]]
+| style="width:350px;border:1px solid black;" |[[File:Psgel11208.jpg]]
 | style="width:350px;border:1px solid black;vertical-align:top;" |
-*Well 1 : 6µL DNA Ladder
+* Well 1 : 6µL DNA Ladder
-*Well 2 : 5µL of digested Bba_K1155000+1µl of 6X loading dy
+* Well 2 : 5µL BBa_K1155000 digested by SpeI/PstI + 1µl of 6X loading dye
-*Well 3 : 5µL of digested Bba_K1155007+1µl of 6X loading dy
+* Well 3 : 5µL BBa_K1155007 digested by XBaI/PstI + 1µl of 6X loading dye
-*Well 4 : 5µL of digested Bba_K1155003+1µl of 6X loading dy
+* Well 4 : 5µL BBa_K1155003 digested by xBaI/PstI + 1µl of 6X loading dye
-*Gel : 0.8%
+* Gel : 0.8%
 |}
 Expected sizes :
-*RBS_LacZ_Term : 3500 kb
+* Pndh* : 111bp
-*RBS_AmilCP_Term :
+* RBS_LacZ_Term : 3500 kb
-*PSB1C3 :
+* RBS_AmilCP_Term : 824 bp
+* pSB1C3 : 2070bp
- We can't see any band for Bba_K1155000 digestion. We just do it again and let it in the fridge over night.
+{|
-...
+| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
+We can't see any band for BBa_K1155000 digestion. The digestion failed. We will do it again. We obtain RBS-LacZ-Term and RBS-AmilCP-Term fragments. The digestion was good. We will purify it.
+|}
-===='''1 - Digestion of Bba_K1155000, Bba_K11'''====
+===='''3 - Digestion of BBa_K1155000 by Spe I/Pst I'''====
-Abdou
-Clone 10,14 and 15 plamid extraction using nucleospin kit.
+Anaïs, Nadia
+Used quantities :
-Protocol : [[Team:Paris_Saclay/Protocols/hight copy plamid extraction|hight copy plamid extraction]]
+* Buffer FD : 5µL
+* H2O : 38µL
+* DNA : 5µL
+* SpeI FD : 1µL
+* PstI FD : 1µL
-Results: concentration measured by nanodrop
+We incubate the digestion at 37°C during 15 minutes.
-*clone 10: C=38ng/µl   260/280= 1.78
-*clone 14: C=48.5ng/µl   260/280=1.90
-*clone 15: C=52 ng/µl     260/280=1.78
-We still have to sequence plasmids in order to verify our results.
+==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
+===='''Objective : obtaining FNR and BphR2 proteins'''====
+===='''1 - Electrophoresis of PCR products : RBS-BphR2 Part I, BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I and FNR Part II '''====
+Damir
+{|
+| style="width:350px;border:1px solid black;" |[[File:Psgel21208.jpg]]
+| style="width:350px;border:1px solid black;vertical-align:top;" |
+* Well 1 : 6µL DNA Ladder
+* Well 2 : 5µL RBS-BphR2 Part I +1µl of 6X loading dye
+* Well 3 : 5µL BphR2 Part II +1µl of 6X loading dye
+* Well 4 : 5µL FNR Part I +1µl of 6X loading dye
+* Well 5 : 5µL FNR Part II +1µl of 6X loading dye
+* Well 6 : 5µL RBS-FNR Part I +1µl of 6X loading dye
+* Well 7 : 5µL BphR2 Part I +1µl of 6X loading dye
+* Gel : 0.8%
+|}
+Expected size
+* RBS-BphR2 Part I : 197 kb
+* BphR2 Part II : 790 kb
+* FNR Part I : 597 kb
+* FNR Part II : 200 kb
+* RBS-FNR Part I : 615 kb
+* BphR2 Part I : 178 kb
+{|
+| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
+We can't see FNR Part I, FNR Part II and BphR2 Part I fragments at the good size. We will make the PCR again. We obtain RBS-BphR2 Part I, BphR2 Part II, RBS-FNR Part I frangments at the right size thanks to the PCR. We will purify it.
+|}
+{| border="1" align="center"
+|[[Team:Paris Saclay/Notebook/August/9|<big>Previous day</big>]]
+|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
+|[[Team:Paris Saclay/Notebook/August/13|<big>Next day</big>]]
+|}
 {{Team:Paris_Saclay/incl_fin}}

Team:Paris Saclay/Notebook/August/12

From 2013.igem.org