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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 | + | ===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''==== |
| - | ===='''1 - Plasmid extraction of | + | ===='''1 - Plasmid extraction of BBa_K1155000 from DH5α'''==== |
Nguyen | Nguyen | ||
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Nanodrop : | Nanodrop : | ||
| - | * | + | * BBa_K1155000 : 175ng/µL |
{| | {| | ||
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|} | |} | ||
| - | ===='''2 - Digestion of | + | ===='''2 - Digestion of BBa_K1155000 by SpeI'''==== |
Nguyen | Nguyen | ||
Used quantities : | Used quantities : | ||
| - | * | + | * BBa_K1155000 : 10µL |
* Buffer FD : 2µL | * Buffer FD : 2µL | ||
* Spe I : 2µL | * Spe I : 2µL | ||
| Line 37: | Line 37: | ||
===='''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 | + | ===='''1 - Gel purification of the digestion of BBa_J04450 by EcoRI/PstI '''==== |
XiaoJing | XiaoJing | ||
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|} | |} | ||
| - | ===='''2 - Electrophoresis of gel purification of the digestion of | + | ===='''2 - Electrophoresis of gel purification of the digestion of BBa_J04450 by EcoRI/PstI '''==== |
XiaoJing | XiaoJing | ||
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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 of | + | * Well 2 : 5µL of BBa_J04450 digested by EcoRI/PstI+1µl of 6X loading dye |
* Gel : 1% | * Gel : 1% | ||
|} | |} | ||
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|} | |} | ||
| - | ===='''3 - Ethanol precipitation of the digestion of | + | ===='''3 - Ethanol precipitation of the digestion of BBa_J04450 by EcoRI/PstI '''==== |
Protocol : [[Team:Paris_Saclay/ethanol|EtOH precipitation]] | Protocol : [[Team:Paris_Saclay/ethanol|EtOH precipitation]] | ||
Revision as of 09:02, 30 September 2013
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
Nguyen
Used quantities :
- BBa_K1155000 : 10µL
- Buffer FD : 2µL
- Spe I : 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
| ]] |
|
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
- DMS9 : 2µL ??????????????????????????????????
- H2O : 31µL
PCR program :
2 - Electrophoresis of PCR product : BphR2 Part I
Damir
| ]] |
|
Expected sizes : Bphr2 Part I : 178 kb
|
We obtain 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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