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Team:Paris Saclay/Notebook/August/8
From 2013.igem.org
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| - | *6µL DNA Ladder | + | * Well 1 : 6µL DNA Ladder |
| - | *10µL | + | * Well 2 to 24 : 10µL of Bba_K1155007+2µL of 6X loading dye |
| + | * Well 25 : 6µL DNA Ladder | ||
| + | * Well 26 : 6µL DNA Ladder | ||
| + | * Well 27 : 10µL of Bba_K1155007+2µL of 6X loading dye | ||
*Gel : 0.8% | *Gel : 0.8% | ||
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| - | Expected size : | + | Expected size : |
| + | * Bba_K1155007 : 3583 bp | ||
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| + | {| | ||
| + | | style="border:1px solid black;padding:5px;background-color:#DEDEDE;" | | ||
| + | We obtain fragment at the right size for colonies 10, 14 and 15. We will extract BBa_K1155007. | ||
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| + | ==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''=== | ||
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| + | ===='''Objective : obtaining FNR and BphR2 proteins'''==== | ||
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====3 - PCR product (made the 08/01/2013) purification==== | ====3 - PCR product (made the 08/01/2013) purification==== | ||
Revision as of 17:21, 16 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 was right
Damir, Nadia
| IMAGE |
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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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Now we can do a gel purification of 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
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
|
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
3 - PCR product (made the 08/01/2013) purification
Damir
available quantity:
- FNR Part1 : 10 µl
- FNR Part2 : 19 µl
- RBS FNR Part1 :16.1µl
- RBS BphR2 Part1 : 28µl
- BphR2 Part1 : 16.4 µl
- BphR2 Part2 : 18.9 µl
Protocol : kit purification
Manipulation error : The elution step was made using the recuperation tube from the filtering step, instead of a new, clean eppendorf tube.
