Team:Paris/14 August 2009

Digestion of pSB1A3 w/: - EcoRI/PstI - EcoRI/SpeI - XbaI/PstI

Mix

DNA : 10 uL

10X Buffer : 3 uL

Enz1 : 1 uL

Enz2 : 1uL

BSA : 0,5 uL

H2O : 14,5 uL

Digestion of:

ClyA(A10) in X/P---->D30

PSB2K3(P27) in X/P-->D31

using:

H2O : 20,5µL

DNA : 20µL

BSA : 0,5µL

Buffer 2(x10) : 5µL

Xba : 2µL

Pst : 2µL

Ligation of ClyA C-ter fusion cut in X/P (D31) in PSB1A3 cut in X/P (P25)

DO insert D31(ClyA C-ter fusion):2µg/mL (1/100) DO vector P25(PSB1A3):0,60µg/mL (1/100)

Insert is two times smaller than the vector so we have to put 2times more insert.

x10 mix: 1µL vector

(2*10)20µL insert

2,5µL buffer 10x

1µL ligase

2µL H2O

x3 mix: 1µL vector

(2x3)6µL insert

1µL buffer 10X

1µL ligase

1µL H2O

negative control: 1µL vector

1µL buffer 10x

1µL ligase

7µL H2O

Then tranformation by Guillaume