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Team:Paris Saclay/Notebook/August/1
From 2013.igem.org
(Difference between revisions)
(→1 - Gel purification of PCR products : BphR2 Part I, BphR2 Part II, FNR Part I, FNR Part II, RBS-FNR Part I in plasmid pSB1C3) |
(→2 - Gibson assembly.) |
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| Line 35: | Line 35: | ||
* BphR2 : | * BphR2 : | ||
| - | ** | + | ** Gibson PCR of pSB1C3 : 2µL |
** BphR2 Part I : 1µL | ** BphR2 Part I : 1µL | ||
** BphR2 Part II : 1µL | ** BphR2 Part II : 1µL | ||
| Line 42: | Line 42: | ||
* FNR : | * FNR : | ||
| - | ** | + | ** Gibson PCR of pSB1C3 : 2µL |
** FNR Part I : 1µL | ** FNR Part I : 1µL | ||
** FNR Part II : 1µL | ** FNR Part II : 1µL | ||
| Line 49: | Line 49: | ||
* RBS-FNR : | * RBS-FNR : | ||
| - | ** | + | ** Gibson PCR of pSB1C3: 2µL |
** RBS-FNR Part I : 1µL | ** RBS-FNR Part I : 1µL | ||
** FNR Part II : 1µL | ** FNR Part II : 1µL | ||
| Line 55: | Line 55: | ||
** H2O : 1µL | ** H2O : 1µL | ||
| - | We | + | We incubated these Gibson assembly mix at 50°C during 1h inside PCR machine. |
===='''3 - PCR of : RBS-BphR2 Part I'''==== | ===='''3 - PCR of : RBS-BphR2 Part I'''==== | ||
Revision as of 16:45, 3 October 2013
Notebook : August 1
Lab work
A - Aerobic/Anaerobic regulation system / B - PCB sensing system
Objective : obtaining FNR and BphR2 proteins
1 - Gel purification of PCR products : BphR2 Part I, BphR2 Part II, FNR Part I, FNR Part II, RBS-FNR Part I in plasmid pSB1C3
Xavier
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
Nanodrop :
- BphR2 Part I : 44 ng/µL
- BphR2 Part II : 64 ng/µL
- FNR Part I : 147 ng/µL
- FNR Part II : 140 ng/µL
- RBS-FNR Part I : 167 ng/µL
- PSB1C3 : 159 ng/µL
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The PCR products were good. Now we will do the Gibson assembly. |
2 - Gibson assembly.
Abdou, Xiaojing
Used quantities :
- BphR2 :
- Gibson PCR of pSB1C3 : 2µL
- BphR2 Part I : 1µL
- BphR2 Part II : 1µL
- Gibson mix : 15µL
- H2O : 1µL
- FNR :
- Gibson PCR of pSB1C3 : 2µL
- FNR Part I : 1µL
- FNR Part II : 1µL
- Gisbon mix : 15µL
- H2O : 1µL
- RBS-FNR :
- Gibson PCR of pSB1C3: 2µL
- RBS-FNR Part I : 1µL
- FNR Part II : 1µL
- Gibson mix : 15µL
- H2O : 1µL
We incubated these Gibson assembly mix at 50°C during 1h inside PCR machine.
3 - PCR of : RBS-BphR2 Part I
Abdou
Used quantities :
- Oligo 54F : 1µL
- Oligo 55R : 1µL
- Buffer phusion : 5µL
- DNA : 0.25µL
- dNTP : 1µL
- Phusion : 5µL
- H2O : 36.5µL
PCR Program :
3 - Electrophoresis of the PCR products : RBS-BphR2 Part I
Xavier, XiaoJing
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Expected sizes :
- RBS-BphR2 Part I : 197pb
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We obtain fragments at the right size. We will purify it. |
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