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Team:Paris/10 August 2009
From 2009.igem.org
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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 |
