Team:Paris/14 August 2009
From 2009.igem.org
NoteBook
|
|
|
|
|
---|
Lab work
Digestion/Gel purification
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/Purification
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/Transformation
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
To do list
Matricule | TODO |
Luc | |
Romain | |
Charlotte | |
Stoff | |
Chris | |
Lisa | |
Caroline | |
Souf | |
Vicard | |
Pierre | |
Sylvain | New Glycerol stocks, OmpA-Linker : new PCR, test the new clone miniprep (Charlotte) with purif |
Guillaume |