Biology Reference
In-Depth Information
3
Methods
3.1 First Targeting
to Generate the
Recombineering
Intermediate
1. Transform
E. coli
strain SW102 with the target Ad-BAC by
electroporation (
see
Note 1
), incubate transformants on selec-
tive LB plates, isolate single colonies and test them for integrity
(
see
Note 2
). Alternatively, plate out frozen stocks of SW102
cells already transformed with the target BAC on selective LB
plates.
2. Design homology-fl anked primers for amplifi cation of
galK-
kan
cassette of pgalK-Kn (
see
Fig.
1b
for details of primer
design and Fig.
1c
for pgalK-Kn map). Insert the 50 nucleo-
tide (nt) target sequence from the upper strand, which is
directly upstream to the sequence to be modifi ed (replaced or
deleted), at the 5
′
end of the forward priming site
(5
). In the same
way, insert the downstream 50 nt homology from the comple-
mentary strand to the 5
′
-CCTGTTGACAATTAATCATCGGCA-3
′
′
end of the reverse primer
) (
see
Note 3
).
3. Perform PCR on 5 ng of pgalK-Kn template using the
homology-fl anked primers by a proofreading polymerase
(
see
Note 4
).
4. Digest the PCR product by
Dpn
I to remove template mole-
cules from the reaction (
see
Note 5
). Set up the digestion reac-
tion using the entire volume of the PCR diluted at least three
times (normally the fi nal volume of the digestion reaction is
300
(5
′
-GCCAGTGTTACAACCAATTAACC-3
′
μ
L for a 100
μ
L PCR reaction). Add 60 U (20 U for
L of fi nal volume) of
Dpn
I and run the reaction over-
night at 37 °C.
5. Purify the
Dpn
I-treated PCR product by any commercial
clean-up columns and verify its integrity by agarose gel
electrophoresis.
6. Inoculate 5 mL LB containing 25
100
μ
g/mL chloramphenicol
with 3-4 colonies of the transformed/plated SW102 contain-
ing the desired target BAC. Incubate at 32 °C overnight in a
shaking incubator.
7. Add 500
μ
L of overnight culture into each of two culture fl asks
with 25 mL LB containing 25
μ
g/mL chloramphenicol and
continue the incubation at 32 °C in a shaking incubator. In
parallel turn on the shaking water bath at 42 °C.
8. Incubate the 25 mL cultures at 32 °C until they reach OD
600 nm
of 0.55-0.6 (takes approximately 3 h). Then, transfer the fl ask
to the shaking water bath and heat shock the cultures at 42 °C
for exactly 15 min (
see
Note 6
).
μ
