Team:Paris/11 August 2009

NoteBook
August 11th  

Brain work
edit please ^^

 

Lab work
Digestion A11 (OmpAsignal) and A13 (TolRII) 2h 37°C

20min 65°C (stop enzymes) purification A11 (OmpAsignal) and A13 (TolRII) after digestion

promega kits

50µl

now A11=D13 A13=D14

Ligation D13 (OmpAsignal X/P) and D14 (TolRII X/P) and vecteur pSB1A3 digested with XbaI and PstI

DO : D13=0,94  D14=1,29   pSB1A3=0,74

1h Home temperature

Transformation classic protocol with DH5a

Ligation D11 (RBS-Tet digested by Xba/Pst) and vecteur D12 (pSB2K3 w/ pLac digested by Spe/Pst)

DO : D11=  D12=

PCR
 * For A10(ClyA) because we have 2 bands in the last gel migration of A3(ClyA) purification (06/08/09)
 * without DMSO
 * with [010] and [031]
 * 1:98°C 1min
 * 2:98°C 10s
 * 3:55°C, 60°C, and 65°C 30s
 * 4:72°C 30s
 * 5:72°C 10min
 * 6: 4°C ~


 * Gel-->[Ladder 1kb/A10 at 55°C/A10 at 60°C/A10 at 65°C]
 * A10 at 55°C work the best we have the biggest and brightest band with this temperature.

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


 * Gel-->[Ladder 100pb/D15/Ladder 1kb/D16]
 * (15min migration) [[Image:Digestion_1.JPG]]
 * (35min migration) [[Image:Digestion_2.JPG]]

All Manip Comming soon

 