Transcript Scheme of procedures for C. elegans transgene construction by fosmid DNA recombineering Original RT recombineering Vectors Culture of Fosmid clone of Gene of Interest.

Scheme of procedures for C. elegans transgene construction by
fosmid DNA recombineering
Original RT recombineering
Vectors
Culture of Fosmid clone of Gene of Interest (G.O.I)
50 bp homology arm of G.O.I
FLAG
N-end(50bp)GFP
Galk
C-end(50bp)GFP
pMOD4
ompF
pBAC-RT
pMOD4GalK-G (3462 bp)
rpsL
TetA
Extraction of G.O.I Fosmid DNAs
2d
1381bp cassette for GalK recombineering
Transformation of G.O.I Fosmid DNAs into SW106
50 bp homology arm of G.O.I
High fidelity PCR, Dpn I digestion and gel–recovery
FLAG
N-end(50bp)GFP
ompF
rpsL
TetA
C-end(50bp)GFP
pMOD4
50 bp of G.O.I
ompF rpsL
rpsL
TetA
TetA
Preparation of SW106 Competent cells of G.O.I
Fosmid DNAs with induction in water bath
50 bp of G.O.I
pMOD4 RT-G (4615 bp)
2548bp cassette for RT recombineering
G.O.I
2.5
d
Lox P
1st recombineering with RT positive selection (Tet+Chl+)
N-end(50bp)NTAP
N-end(50bp)CTAP
FLAG
N-end(50bp)GFP
Galk
C-end(50bp)GFP
C-end(50bp) CTAP
c-end(50bp)NTAP
50 bp homology arm of G.O.I
TAG ( GFP or TAP)
pMOD4
pMOD4GalK-GT (3693 bp)
G.O.I
ompF rpsL
rpsL
TetA
TetA
G.O.I
Lox P
1612 bp cassette for GalK recombineering
50 bp homology arm of G.O.I
PCR and gel-recovery
FLAG
GFP
3.5
d
pMOD4
Preparation of Competent cells
2nd recombineering with RT negative selection on
hypotonic media (Str+Chl+)
pMOD4-FLAG-GFP ( 4029bp)
50 bp of G.O.I
G.O. I
Lox P
UNC-119
pMOD4)
TAG ( GFP or TAP)
TAG (GFP or TAP)
50 bp of G.O.I
G.O.I
Lox P
pLoxP-UNC-119 ( 7846bp)
Modified RT recombineering
gal K recombineering
50 bp homology arm of G.O.I
50 bp homology arm of G.O.I
Culture of Fosmid clone of Gene of Interest (G.O.I)
GalK
pMOD4GalK-GT
ompF rpsL
pMOD4RT-G
pMOD4
Culture of Fosmid clone of Gene of Interest (G.O.I)
rpsL TetA
TetA
pMOD4
Extraction of G.O.I Fosmid DNAs
Extraction of G.O.I Fosmid DNAs
2d
50 bp homology arm of G.O.I
2d
High fidelity PCR and gel–recovery
Transformation of G.O.I Fosmid DNAs into SW106
50 bp homology arm of G.O.I
High fidelity PCR and gel–recovery
50 bp of G.O.I
50 bp of G.O.I
GalK
ompF rpsL
rpsL TetA
TetA
Preparation of SW106 Competent cells of G.O.I
Fosmid DNAs with induction in water bath
50 bp of G.O.I
Preparation of SW106 Competent cells of G.O.I
Fosmid DNAs with induction in water bath
50 bp of G.O.I
Transformation of G.O.I Fosmid DNAs into SW106
G.O.I
G.O.I
3.5 d
Lox P
2.5
d
Lox P
1st recombineering with RT positive selection (Tet+Chl+)
1st recombineering with galactose positive selection (Chl+)
GFP
TAG (GFP or TAP)
G.O.I
G.O.I
GalK
ompF rpsL
rpsL TetA
TetA
G.O.I
G.O.I
Lox P
Lox P
Verifying on MacConkey Agar individually
Preparation of Competent cells
recombineering with 2-Deoxy-galactose negative selection
(Chl+)
G.O.I
50 bp of G.O.I
TAG (GFP or TAP)
TAG (GFP or TAP)
Preparation of Competent cells
2nd recombineering with RT negative on
hypotonic media (Str+Chl+)
50 bp of G.O.I
UNC-119
pMOD4
pLoxP-UNC-119
ampR
TAG ( GFP or TAP)
G.O.I
UNC-119
50 bp of G.O.I
G.O.I
+
Lox P
2d
pLoxP / Cre recombineering with 0.1% arabinose (AMP+Chl+)
TAG ( GFP or TAP)
TAG ( GFP or TAP)
Lox P
+
Lox P
50 bp of G.O.I
G.O. I
G.O.I
Lox P
G.O. I
3.5
d
5d
2nd
Lox P
or
or
UNC-119
pMOD4
pLoxP-UNC-119
2d
ampR
pLoxP / Cre recombineering with 0.1% arabinose (AMP+Chl+)
G.O. I
ampR
Lox P
TAG ( GFP or TAP)
G.O.I
UNC-119
ampR