"
Team:Paris/10 August 2009
From 2009.igem.org
(Difference between revisions)
(→Lab work) |
(→Lab work) |
||
| (12 intermediate revisions not shown) | |||
| Line 31: | Line 31: | ||
</div> | </div> | ||
<div class="experience"> | <div class="experience"> | ||
| - | *For [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A8 A8]OmpA-Linker and [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A9 A9]pFecA | + | *For [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A8 A8] OmpA-Linker and [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A9 A9] pFecA |
*Duplicate with and without DMSO | *Duplicate with and without DMSO | ||
**1:98°C 1min | **1:98°C 1min | ||
| Line 141: | Line 141: | ||
<br> | <br> | ||
*Ligation 1 | *Ligation 1 | ||
| - | **10µl RBS- | + | **10µl RBS-tetR XbaI/PstI |
**1µl pSB2K3(pLac) SpeI/PstI | **1µl pSB2K3(pLac) SpeI/PstI | ||
**2µl Buffer T4 DNA Lygase 10x | **2µl Buffer T4 DNA Lygase 10x | ||
| Line 148: | Line 148: | ||
<br> | <br> | ||
*10min at room temperature (~25?) | *10min at room temperature (~25?) | ||
| + | *20min at 80°C | ||
</div> | </div> | ||
<div class="guillaume"> | <div class="guillaume"> | ||
| Line 160: | Line 161: | ||
</div> | </div> | ||
<div class="experience"> | <div class="experience"> | ||
| - | [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A11 A11](OmpAsignal)[https://2009.igem.org/Team:Paris/Freezer_PCR_products#A12 A12](Tg3p)[https://2009.igem.org/Team:Paris/Freezer_PCR_products#A13 A13](TolRII) without (1,2,3) and with DMSO (4,5,6) | + | [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A11 A11] (OmpAsignal) [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A12 A12] (Tg3p) [https://2009.igem.org/Team:Paris/Freezer_PCR_products#A13 A13] (TolRII) without (1,2,3) and with DMSO (4,5,6) |
temperature gradient | temperature gradient | ||
| Line 171: | Line 172: | ||
A13 : 71,8 : 73,1 : 74,0 | A13 : 71,8 : 73,1 : 74,0 | ||
| + | |||
| + | Waiting Heigh = A11: 135pb, A12: 274pb, A13: 279pb | ||
->gel | ->gel | ||
| - | A11 and A13 good | + | [[Image:OmpAsignal_fusion_Nterm.JPG]] |
| + | |||
| + | |||
| + | [[Image:Tg3p_fusion_N_term.JPG]] | ||
| + | |||
| + | |||
| + | [[Image:TolRII fusion N term.JPG]] | ||
| + | |||
| + | '''A11(OmpAsignal) and A13(TolRII) good and nothing for A12(Tg3p)''' | ||
</div> | </div> | ||
| Line 182: | Line 193: | ||
<div class="experience"> | <div class="experience"> | ||
purification on tube n°5 (A11.5 : T°: 70,2 with DMSO and A13.5 : T°73,1 with DMSO)with promega kits | purification on tube n°5 (A11.5 : T°: 70,2 with DMSO and A13.5 : T°73,1 with DMSO)with promega kits | ||
| - | gel purification -> Very good | + | |
| + | 50µl final | ||
| + | |||
| + | gel purification -> '''Very good''' | ||
| + | |||
| + | [[Image:purif OmpA signal fusion & tolRII fusion N term.JPG]] | ||
| + | |||
| + | Next : Digestion tomorrow | ||
</div> | </div> | ||
<html> | <html> | ||
Latest revision as of 11:42, 11 August 2009
Contents |
NoteBook
|
|
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Brain work
edit please ^^
Lab work
PCR
Mini-Prep for Plac
Same Protocol as usual ...
Do for sequencing
| Dilution for Plasmids 1,2&3 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Plasmid | D0(000) | D0(266/280) | D0(320) | Dilution Rule | Primer Addition | Water Addition | ||||
| P1 | 3.10 | 1.72 | 0.004 | 600/310 = 1.93 ~ 2.0 µL | 2.45 µL | 10.55 µL | ||||
| P2 | 1.34 | 1.34 | 0.000 | 600/134 = 4.48 (~ 4.5 µL) | 2.45 µL | 8.05 µL | ||||
| P3 | 0.70 | 3.39 | 0.000 | 600/70 = 8.57 ~ 8.6 µL | 2.45µL | 3.95µL | ||||
Gel Migration
GEL!!!
- Remigrate to be sure... pFecA seem to be good but not Ompa-Linker
Digestion
- Digestion of P21 (pLac in pSB2K3) with SpeI/PstI (D12) in order to insert D11 RBS-tet (XbaI/PstI)
- 20µl of P21
- Vfinal=50µl
- 2h at 37°C
Purification
- Promega kit
- 50µl final
GEL!!!(strange! none solution fall as quickly in the wholes)
DO260
- D11: RBS-tet : 0,10µg/ml ~730bp
- D12: pSB2K3(pLac) : 0,96µl/ml ~4400bp
Try for ligation
- Ligation between [D12] pSB2K3(pLac) SpeI/PstI and [D11] RBS-tet XbaI/PstI. 20µl final
- Ligation 1
- 4,5µl RBS-tet XbaI/PstI
- 1µl pSB2K3(pLac) SpeI/PstI
- 2µl Buffer T4 DNA Lygase 10x
- 1µl T4 DNA Lygase
- 11,5µl H20
- Ligation 1
- 10µl RBS-tetR XbaI/PstI
- 1µl pSB2K3(pLac) SpeI/PstI
- 2µl Buffer T4 DNA Lygase 10x
- 1µl T4 DNA Lygase
- 6µl H20
- 10min at room temperature (~25?)
- 20min at 80°C
Transformation of the ligation
- As usual, but I forget to done transformation with vector only...so I haven't any control... --!
PCR
A11 (OmpAsignal) A12 (Tg3p) A13 (TolRII) without (1,2,3) and with DMSO (4,5,6)
temperature gradient
tube n°1/4 : 2/5 : 3/6
A11 : 68,6 : 70,2 : 71,8
A12 : 62,2 : 62,9 : 64,0
A13 : 71,8 : 73,1 : 74,0
Waiting Heigh = A11: 135pb, A12: 274pb, A13: 279pb
->gel
A11(OmpAsignal) and A13(TolRII) good and nothing for A12(Tg3p)
Purification
purification on tube n°5 (A11.5 : T°: 70,2 with DMSO and A13.5 : T°73,1 with DMSO)with promega kits
50µl final
gel purification -> Very good
Next : Digestion tomorrow
To do list
| Matricule | TODO |
| Luc | |
| Romain | |
| Charlotte | |
| Stoff | |
| Chris | |
| Lisa | |
| Caroline | |
| Souf | |
| Vicard | |
| Pierre | modelisation & Algorithmic (a bit...) |
| Sylvain | |
| Guillaume |
