"
Team:Paris Saclay/Notebook/August/8
From 2013.igem.org
CarolineMir (Talk | contribs) (→1 - Tranduction of Km in MG1655Z1) |
|||
| Line 9: | Line 9: | ||
===='''Obtaining biobricks in PSB3K3'''==== | ===='''Obtaining biobricks in PSB3K3'''==== | ||
| - | ====1 - Digestion of | + | ====1 - Digestion of BBa_J04450 by EcoRI/PstI==== |
Nadia | Nadia | ||
| Line 43: | Line 43: | ||
|} | |} | ||
| - | ====3 - Electrophoresis of | + | ====3 - Electrophoresis of BBa_J004450 digested by EcoRI/PstI==== |
Anaïs | Anaïs | ||
| Line 71: | Line 71: | ||
|} | |} | ||
| - | ===='''Objective : obtaining | + | ===='''Objective : obtaining BBa_K1155007'''==== |
| - | ====1 - Colony PCR of | + | ====1 - Colony PCR of BBa_K115007 in DH5α==== |
Anaïs | Anaïs | ||
| Line 98: | Line 98: | ||
[[File:PsPcr808.jpg|400px]] | [[File:PsPcr808.jpg|400px]] | ||
| - | ====2 - Electrophoresis to check the colony PCR products : | + | ====2 - Electrophoresis to check the colony PCR products : BBa_K1155007==== |
Anaïs, Damir | Anaïs, Damir | ||
| Line 106: | Line 106: | ||
| style="width:350px;border:1px solid black;vertical-align:top;" | | | style="width:350px;border:1px solid black;vertical-align:top;" | | ||
* Well 1 : 6µL DNA Ladder | * Well 1 : 6µL DNA Ladder | ||
| - | * Well 2 to 24 : 10µL of | + | * Well 2 to 24 : 10µL of BBa_K1155007+2µL of 6X loading dye |
* Well 25 : 6µL DNA Ladder | * Well 25 : 6µL DNA Ladder | ||
* Well 26 : 6µL DNA Ladder | * Well 26 : 6µL DNA Ladder | ||
| - | * Well 27 : 10µL of | + | * Well 27 : 10µL of BBa_K1155007+2µL of 6X loading dye |
*Gel : 0.8% | *Gel : 0.8% | ||
|} | |} | ||
Expected size : | Expected size : | ||
| - | * | + | * BBa_K1155007 : 3583 bp |
{| | {| | ||
| Line 121: | Line 121: | ||
|} | |} | ||
| - | ===='''Objective : characterize | + | ===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''==== |
===='''1 - Tranduction of Km in MG1655Z1 ==== | ===='''1 - Tranduction of Km in MG1655Z1 ==== | ||
Revision as of 09:22, 30 September 2013
Notebook : August 8
Lab work
A - Aerobic/Anaerobic regulation system
Obtaining biobricks in PSB3K3
1 - Digestion of BBa_J04450 by EcoRI/PstI
Nadia
Used quantities :
- DNA : 5µL
- Buffer FD : 2µL
- EcoRI FD : 1µL
- PstI FD : 1µL
- H2O : 11µL
We let our digestion 1h30 at 37°C.
2 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion
Damir, Nadia
| IMAGE |
|
Expected sizes :
- PSB3K3 : 2750kb
- GFP : 1069kb
|
We obtain fragments at the right size. We will purify the highest band which contains the PSB3K3 plasmid. |
3 - Electrophoresis of BBa_J004450 digested by EcoRI/PstI
Anaïs
|
|
|
We obtain fragments at the right size. We will purify the highest band which contains the PSB3K3 plasmid. |
4- Electroelution of PSB3K3 digested by EcoRI/PstI
Nadia
Protocol : Electroelution
|
We lost our plasmid. We will do the digestion again. |
Objective : obtaining BBa_K1155007
1 - Colony PCR of BBa_K115007 in DH5α
Anaïs
|
Tranformation of 08/07/13 works. we will make a PCR Colony. |
We mixed our colonies in 10µL of H2O.
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
| 350px |
|
Expected size :
- BBa_K1155007 : 3583 bp
|
We obtain fragment at the right size for colonies 10, 14 and 15. We will extract BBa_K1155007. |
Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006
1 - Tranduction of Km in MG1655Z1
Anaïs, Nadia
|
We didn't obtain colonies from the transduction of 08/07/13. We will do it again. |
Protocol : Transduction
Our Mutant bacteria was called BW : Δfnr::Km. Our wild type bacteria was called MG1655Z1.
We did the first step of the protocol : bacteriophage stock which packed Km gene.
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
|
Transformation of 08/02/13 works. We will extract plasmids. |
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
|
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 ]
|
We lost fragment. We will do the PCR again. |
| Previous day | Back to calendar | Next day |
