Team:Paris/11 August 2009

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← Yesterday - Tomorrow →

August 11th

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

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

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 - o 2µl o 15µl 2µl 1µl 20µl


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=

vecteur insert H20 Buffer 10x T4 ligase T4 ligase Total
L1 D11 µl µl µl µl µl µl
control - o µl o µl µl µl µl


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]
ClyA A10.JPG
  • 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)
    Digestion 1.JPG
  • (35min migration)
    Digestion 2.JPG

All Manip

Comming soon

To do list

Matricule TODO
Luc
Romain
Charlotte
Stoff
Chris
Lisa
Caroline
Souf
Vicard
Pierre
Sylvain
Guillaume


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