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Team:Paris Saclay/Notebook/August/12
From 2013.igem.org
(Difference between revisions)
(→3 - Digestion of BBa_K1155000 by SpeI/PstI) |
(→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) |
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** PstI FD : 1µL | ** PstI FD : 1µL | ||
| - | We | + | 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'''==== | ===='''2 - Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBalI/PstI'''==== | ||
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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 DNA Ladder | * Well 1 : 6µL DNA Ladder | ||
| - | * Well 2 : 5µL BBa_K1155000 digested by | + | * Well 2 : 5µL BBa_K1155000 digested by SpeI/PstI + 1µl of 6X loading dye |
| - | * Well 3 : 5µL BBa_K1155007 digested by | + | * Well 3 : 5µL BBa_K1155007 digested by XBaI/PstI + 1µl of 6X loading dye |
| - | * Well 4 : 5µL BBa_K1155003 digested by | + | * Well 4 : 5µL BBa_K1155003 digested by xBaI/PstI + 1µl of 6X loading dye |
* Gel : 0.8% | * Gel : 0.8% | ||
|} | |} | ||
Expected sizes : | Expected sizes : | ||
| - | * | + | * Pndh* : 111bp |
* RBS_LacZ_Term : 3500 kb | * RBS_LacZ_Term : 3500 kb | ||
| - | * RBS_AmilCP_Term : | + | * RBS_AmilCP_Term : 824 bp |
* pSB1C3 : 2070bp | * pSB1C3 : 2070bp | ||
| Line 72: | Line 72: | ||
* H2O : 38µL | * H2O : 38µL | ||
* DNA : 5µL | * DNA : 5µL | ||
| - | * | + | * SpeI FD : 1µL |
| - | * | + | * PstI FD : 1µL |
We incubate the digestion at 37°C during 15 minutes. | We incubate the digestion at 37°C during 15 minutes. | ||
| Line 89: | Line 89: | ||
| 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 RBS-BphR2 Part I+1µl of 6X loading dye | + | * Well 2 : 5µL RBS-BphR2 Part I +1µl of 6X loading dye |
| - | * Well 3 : 5µL BphR2 Part II+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 4 : 5µL FNR Part I +1µl of 6X loading dye |
| - | * Well 5 : 5µL FNR Part II+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 6 : 5µL RBS-FNR Part I +1µl of 6X loading dye |
| - | * Well 7 : 5µL BphR2 Part I+1µl of 6X loading dye | + | * Well 7 : 5µL BphR2 Part I +1µl of 6X loading dye |
* Gel : 0.8% | * Gel : 0.8% | ||
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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 can't see FNR Part I, FNR Part II and BphR2 Part | + | 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. |
|} | |} | ||
Latest revision as of 19:26, 3 October 2013
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
|
|
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
|
|
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. |
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