|
|
| Line 17: |
Line 17: |
| | * H20 : 20µL | | * H20 : 20µL |
| | * Buffer FD : 3µL | | * Buffer FD : 3µL |
| - | * | + | * RBS-AmilCP-Term : 5µL |
| | * EcoRI : 1µL | | * EcoRI : 1µL |
| | * PstI : 1µL | | * PstI : 1µL |
| - |
| |
| - | {|
| |
| - | | style="width:350px;border:1px solid black;" | IMAGE
| |
| - | | style="width:350px;border:1px solid black;vertical-align:top;" |
| |
| - | *Well 1 : 6µL DNA Ladder
| |
| - | *Well 2 : 5µL of BBa_J004450 digested by EcoRI/Pst1
| |
| - | *Gel : 0.8%
| |
| - | |}
| |
| - |
| |
| - | 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
| |
| - |
| |
| - | {|
| |
| - | | style="width:350px;border:1px solid black;" | [[File:PsNBa8_eelution.jpg|350px]]
| |
| - | | style="width:350px;border:1px solid black;vertical-align:top;" |
| |
| - | *Well 1 : 6µL DNA Ladder
| |
| - | *Well 2 : 45µL of BBa_J004450 digested by EcoRI/Pst1
| |
| - | *Gel : 0.8%
| |
| - | |}
| |
| - |
| |
| - | 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 : [[Team:Paris_Saclay/Protocols/Electroelution|Electroelution]]
| |
| - |
| |
| - | We let the plasmid precipitate during the night.
| |
| - |
| |
| - | ===='''Obtaining RBS_LacZ+Term_PSB1C3'''====
| |
| - |
| |
| - | =====1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies=====
| |
| - | Anaïs
| |
| - |
| |
| - | *Colony counting :
| |
| - | **Low concentration petri dish : 47 colonies
| |
| - | **High concentration petri dish : 145 colonies
| |
| - |
| |
| - | *Picking of 25 colonies
| |
| - |
| |
| - | *Preparation of 700µL of Master mix
| |
| - | **H<sub>2</sub>O : 590µL
| |
| - | **dNTP : 28µL
| |
| - | **VF2 primer : 3.5µL
| |
| - | **VR primer : 3.5µL
| |
| - | **DreamTaq buffer 10x : 70µL
| |
| - | **DreamTaq enzyme : 5µL
| |
| - |
| |
| - | Protocol : [[Team:Paris_Saclay/Protocols/Colony_PCR|Colony PCR]]
| |
| - |
| |
| - | PCR Program :
| |
| - |
| |
| - | [[File:PsPcr808.jpg|400px]]
| |
| - |
| |
| - | =====2 - Gel electrophoresis of the colony PCR products=====
| |
| - | Anaïs, Damir
| |
| - |
| |
| - | {|
| |
| - | | style="width:350px;border:1px solid black;" | [[File:PsNBa8_colonies.jpg|350px]]
| |
| - | | style="width:350px;border:1px solid black;vertical-align:top;" |
| |
| - | *6µL DNA Ladder
| |
| - | *10µL sample per well
| |
| - | *Gel : 0.8%
| |
| - | |}
| |
| - |
| |
| - | Expected size : 3583bp
| |
| - |
| |
| - | Colonies 10, 14, 15 exhibit plasmids with the right length.
| |
| - |
| |
| - | ====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 : [[Team:Paris_Saclay/Protocols/kit_purification|kit purification]]
| |
| - |
| |
| - | <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.
| |
| - |
| |
| | | | |
| - |
| |
| | | | |
| | | | |
| | | | |
| | {{Team:Paris_Saclay/incl_fin}} | | {{Team:Paris_Saclay/incl_fin}} |
"
