Team:Paris/10 August 2009

NoteBook
August 10th  

Brain work
edit please ^^

 

Lab work
PCR
 * For A8 OmpA-Linker and A9 pFecA
 * Duplicate with and without DMSO
 * 1:98°C 1min
 * 2:98°C 10s
 * 3:59°C for A8) / 65°C for A9 30s
 * 4:72°C 8s
 * 5:72°C 10min
 * 6: 4°C ~

Mini-Prep for Plac Same Protocol as usual ...

Do for sequencing

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 GEL!!!(strange! none solution fall as quickly in the wholes)
 * Promega kit
 * 50µl final

DO260
 * D11: RBS-tet : 0,10µg/ml ~730bp
 * D12: pSB2K3(pLac) : 0,96µl/ml ~4400bp

Try for ligation Transformation of the 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
 * 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  