Team:Paris/11 August 2009
From 2009.igem.org
Contents |
NoteBook
|
|
|
|
|
---|
Brain work
edit please ^^
Lab work
Digestion
A11 (OmpAsignal) and A13 (TolRII)
DNA | Buffer 10X | H20 | BSA | XbaI | PstI | Total |
20µl | 3µl | 4,5µl | 0,5µl | 1µl | 1µl | 30µl |
2h 37°C
20min 65°C (stop enzymes)
purification
Ligation
D13 (OmpAsignal X/P) and D14 (TolRII X/P) and vecteur pSB1A3 digested with XbaI and PstI
DO : A11=0,94 A13=1,29 pSB1A3=0,74
OmpAsignal:pSB1A3
TolRII:pSB1A3
vecteur | insert | H20 | Buffer 10x T4 ligase | T4 ligase | Total | ||
L3 | TolRII:pSB1A3 | 2µl | 3µl | 12µl | 2µl | 1µl | 20µl |
L4 | OmpAsignal:pSB1A3 | 2µl | 3µl | 12µl | 2µl | 1µl | 20µl |
control | 2µl | 12µl | 2µl | 1µl | 20µl |
plus negative control
1h Home temperature
Transformation
classic protocol with DH5a
PCR
Digestion
- Digestion of P4 double terminator with EcoRI/SpeI (D15)in order to ligate with P21 (double terminator)
- 20µl of P21
- 2µl of EcoR1 and 2µl of SpeI
- 0,5µl BSA 100X
- 5µl buffer 2 x10
- 20,5µl H20
- Vfinal=50µl
- 2h at 37°C
- Digestion of P21 (pLac in pSB2K3) with EcoRI/XbaI (D16)in order to ligate with P4 (double terminator)
- 20µl of P21
- 2µl of EcoR1 and 2µl of Xba1
- 0,5µl BSA 100X
- 5µl buffer 2 x10
- 20,5µl H20
- Vfinal=50µl
- 2h at 37°C
To do list
Matricule | TODO |
Luc | |
Romain | |
Charlotte | |
Stoff | |
Chris | |
Lisa | |
Caroline | |
Souf | |
Vicard | |
Pierre | |
Sylvain | |
Guillaume |