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| Line 30: |
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| - | =====2 - Digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI and by SpeI/EcoRI=====
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| | | | |
| - | Anaïs, Nadia
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| | | | |
| - | * Bba_K1155000 :
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| - | ** Buffer : 5µL
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| - | ** H2O : 39µL
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| - | ** DNA : 5µL
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| - | ** SpeI : 1µL
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| | | | |
| - | *Bba_K1155000 :
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| - | ** Buffer : 5µL
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| - | ** H2O : 38µL
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| - | ** DNA : 5µL
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| - | ** SpeI : 1µL
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| - | **EcoRI : 1µL
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| | | | |
| - | *Bba_K1155004, Bba_K1155005, bba_K1155006 :
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| - | ** Buffer : 2µL
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| - | ** H2O : 7µL
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| - | ** DNA : 10µL
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| - | ** SpeI : 1µL
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| - |
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| - | *Bba_K1155004, Bba_K1155005, bba_K1155006 :
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| - | ** Buffer : 2µL
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| - | ** H2O : 6µL
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| - | ** DNA : 10µL
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| - | ** SpeI : 1µL
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| - | ** EcoRI : 1µL
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| - |
| |
| - | ===='''Obtaining RBS_LacZ+Term_PSB1C3'''====
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| - |
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| - | =====1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies=====
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| - | Anaïs
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| - |
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| - | *Colony counting :
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| - | **Low concentration petri dish : 47 colonies
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| - | **High concentration petri dish : 145 colonies
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| - |
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| - | *Picking of 25 colonies
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| - |
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| - | *Preparation of 700µL of Master mix
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| - | **H<sub>2</sub>O : 590µL
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| - | **dNTP : 28µL
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| - | **VF2 primer : 3.5µL
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| - | **VR primer : 3.5µL
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| - | **DreamTaq buffer 10x : 70µL
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| - | **DreamTaq enzyme : 5µL
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| - |
| |
| - | Protocol : [[Team:Paris_Saclay/Protocols/Colony_PCR|Colony PCR]]
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| - |
| |
| - | PCR Program :
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| - |
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| - | [[File:PsPcr808.jpg|400px]]
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| - |
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| - | =====2 - Gel electrophoresis of the colony PCR products=====
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| - | Anaïs, Damir
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| - |
| |
| - | {|
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| - | | style="width:350px;border:1px solid black;" | [[File:PsNBa8_colonies.jpg|350px]]
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| - | | style="width:350px;border:1px solid black;vertical-align:top;" |
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| - | *6µL DNA Ladder
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| - | *10µL sample per well
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| - | *Gel : 0.8%
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| - | |}
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| - |
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| - | Expected size : 3583bp
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| - |
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| - | Colonies 10, 14, 15 exhibit plasmids with the right length.
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| - |
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| - | ====3 - PCR product (made the 08/01/2013) purification====
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| - | Damir
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| - |
| |
| - | available quantity:
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| - | * FNR Part1 : 10 µl
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| - | * FNR Part2 : 19 µl
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| - | * RBS FNR Part1 :16.1µl
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| - | * RBS BphR2 Part1 : 28µl
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| - | * BphR2 Part1 : 16.4 µl
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| - | * BphR2 Part2 : 18.9 µl
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| - |
| |
| - | Protocol : [[Team:Paris_Saclay/Protocols/kit_purification|kit purification]]
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| - |
| |
| - | <span style="color:#FF5500;">Manipulation error :</span> The elution step was made using the recuperation tube from the filtering step, instead of a new, clean eppendorf tube.
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| | | | |
"
