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3. Gateway cloning of TT-PCR products into pDONR207
The Gateway system (Invitrogen) is based on bacteriophage
site-specific recombination
(Landy 1989). Gateway cloning introduces the amplified TT-PCR product into the donor vector,
pDONR207 (Invitrogen), by in vitro recombination between the attB1 and attB2 sequences that flank
the TT-PCR product (see above) and the attP1 and attP2 sequences, respectively, of pDONR207. This
attB x attP recombination is mediated by the BP reaction (Invitrogen) and produces the attL1 and attL2
sequences that flank the tagged gene within the pDONR vector.
Note that unrecombined pDONR vectors should be propagated in the DB3.1 strain of E. coli
(Invitrogen) carrying the gyrA462 gene which confers resistance to the ccdB gene [its protein product, a
natural analog of quinolone antibiotics, binds to the DNA gyrase subunit A and turns it into a poison
(Bahassi et al. 1999)]. Following Gateway recombination, ccdB is replaced by the TT-PCR product,
allowing selection for the recombinant clones in bacterial strains, such as DH5
or DH
, that do not
carry gyrA462 or F’ episome (which also confers resistance to ccdB).
a. BP reaction and selection for recombinant clones
BP reaction mixture
BP reaction conditions
300 ng (in 1-5 µl) TT-PCR product
overnight incubation at 25ºC
150 ng (in 1 µl) pDONR207 (Invitrogen)*
2 µl 5x BP Clonase reaction buffer
2 µl BP Clonase (Invitrogen)
TE buffer (pH 8.0) to total volume of 10 µl
*Note that unrecombined pDONR207 is
toxic to most bacterial strains and
should be propagated in the DB3.1
strain of E. coli (Invitrogen) in the
presence of chloramphenicol and
gentamycin
Add 1 µl Proteinase K (2 µg/µl) and incubate for 10 minutes at 37ºC. Then, transform 2µl of
the reaction mixture into 100 µl competent cells of the E. coli strain DH5
or DH10B and select for
recombinants by plating on LB agar supplemented with 7 µg/ml gentamycin.
b. Identification of recombinant colonies with TT-PCR product
Pick 4 colonies per construct and analyze each by PCR for the presence of the TT-PCR
product. Use the either of the following attL primers in combination with one gene specific primer.
forward attL1 primer: 5’-TCGCGTTAACGCTAGCATGGATCTC-3’
reverse attL2 primer: 5’-GTAACATCAGAGATTTTGAGACAC-3’
PCR reaction mixture
PCR cycles
1 bacterial colony
1x Taq reaction buffer
0.2 mM 4 x dNTP
0.2 µM of each primer
total volume: 20 µl
incubate 10 min at 95°C to release DNA
0.02 U/µl Taq (any brand)
1 cycle:
94°C
3 min
25 cycles:
93°C
30 sec
50°C
30 sec
68°C
1 min per kb
1 cycle:
72°C
1 min per kb
Select positive clones, i.e., those that have the correct size insert, purify their plasmid DNA and
sequence the tagged genes. In our experiments, the efficiency of the recombination of the TT-PCR
products into pDONR207 is 80-90%.
4. Gateway transfer of the tagged genes into binary destination vectors
a. Gateway binary destination vectors
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