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Team:Paris Saclay/Notebook/August/8
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===='''Obtaining the PSB3K3 backbone plasmid'''==== | ===='''Obtaining the PSB3K3 backbone plasmid'''==== | ||
| - | ====1 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion | + | ====1 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion==== |
Damir, Nadia | Damir, Nadia | ||
Revision as of 14:27, 28 September 2013
Notebook : August 8
Lab work
A - Aerobic/Anaerobic regulation system
Obtaining the PSB3K3 backbone plasmid
1 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion
Damir, Nadia
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Expected sizes :
- PSB3K3 : 2750kb
- GFP : 1069kb
We obtained fragments of the right size. Now we can purify the PSB3K3 plasmid.
2 - Gel purification of the PSB3K3 plasmid from BBa_J004450 digested by EcoRI/Pst1
2.1 - Migration of the remaining 45µL of BBa_J004450 digested by EcoRI/Pst1
Anaïs
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We obtain fragments at the right size. We will purify the highest band which contains the PSB3K3 plasmid. |
2.2 - Electroelution of the highest band to extract the PSB3K3 plasmid from the gel
Nadia
Protocol : Electroelution
We let the plasmid precipitate during the night.
Objective : obtaining Bba_K1155007
1 - Colony PCR of Bba_K115007 in DH5α
Anaïs
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Tranformation of 08/07/13 works. we will make a PCR Colony. |
Colonie repiquée dans 10µL d'eau pour chaque tube ???????
Used quantities :
- DNA : 2µL
- Mix : (it was divided in 25 tubes for each promotor with 23µL of mix in each on)
- Oligo ... : 3.5µL
- Oligo ... : 3.5µL
- Buffer Dream Taq : 70µL
- dNTP : 28µL
- Dream Taq : 5µL
- H2O : 590µL
PCR Program :
2 - Electrophoresis to check the colony PCR products : Bba_K1155007
Anaïs, Damir
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Expected size :
- Bba_K1155007 : 3583 bp
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We obtain fragment at the right size for colonies 10, 14 and 15. We will extract BBa_K1155007. |
A - Aerobic/Anaerobic regulation system / B - PCB sensor system
Objective : obtaining FNR and BphR2 proteins
1 - Extraction of plasmid of BphR2, FNR, RBS-FNR
Damir
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Transformation of 08/02/13 works. We will extract plasmids. |
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
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We lost our plasmids. We will do the Gibson assembly again. |
2 - Gel purification of RBS-BphR2 Part I
Nadia, XiaoJing
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
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We lost fragment. We will do the PCR again. |
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