Biology Reference
In-Depth Information
18. Pick 6-12 single pink/red colonies and inoculate 10 mL LB
containing 25
L kana-
mycin. Incubate the cultures at 32 °C overnight in a shaking
incubator. Prepare BAC DNA in small scale using appropriate
purifi cation kit (
see
Note 9
) and subject them to restriction
analysis to confi rm their integrity and save glycerol stocks from
two verifi ed colonies (
see
Note 10
).
μ
g/mL chloramphenicol and 25
μ
g/
μ
1. Synthesize the DNA sequence to be inserted into the target
site either via gene synthesis service or by PCR. Sub-cloned
and modifi ed fragments can also be reintroduced into the BAC
genome after amplifi cation of the desired fragment by PCR.
For this PCR also homology-fl anked primers are designed in
the same way as for the fi rst targeting (
see
Fig.
1b
for details).
Larger fragments derived from viral DNA or even intact
genomic DNA can be introduced into the appropriate recom-
bineering intermediate (
see
Note 11
). The linear fragments
used here need at least 50 nt of homology at each end of the
regions fl anking the galK-Kn cassette of the recombineering
intermediate. If larger DNA fragments are used, longer homol-
ogies may increase the effi ciency.
2. Use two Gal + clones verifi ed by restriction analysis from previ-
ous
step 18
(
see
Note 12
) and inoculate 5 mL LB containing
chloramphenicol and kanamycin and incubate overnight at
32 °C in a shaking incubator.
3. Repeat
steps 7-13
from Subheading
3.1
to obtain electro-
competent bacteria. However, here preparing for the second
targeting, use always LB containing 25
3.2 Second Targeting
for Generating the
Mutants
μ
g/mL chlorampheni-
g/mL kanamycin for all cultures.
4. Transform 60-70
col and 25
μ
L of the induced electrocompetent bacteria
with 200 ng up to 1.5
μ
g of desired linear fragment (
step 1
)
by an electroshock at 2,500 V, 200 W, and 25
μ
F in Gene Pulser
(or equivalent electroporation equipment) (
see
Note 13
).
Prepare the control reaction by electroporating a mixture
containing the same volume of the competent cells in sterile
water and the volume of the DNA fragment. Then, mix
the electroporated bacteria with 1 mL LB without any antibi-
otic and transfer into 50 mL Falcon tubes containing 10 mL
LB without antibiotics. Outgrowth the cultures for 4.5 h at
32 °C in a shaking incubator.
5. Transfer 1 mL culture from each outgrowth to a 2 mL
Eppendorf tube and spin at top speed for 15 s in a microcen-
trifuge at room temperature. Remove the supernatant.
6. Resuspend pellet in 1 mL M9 medium and repeat the centrifu-
gation as in
step 5
and remove the supernatant.
7. Repeat
step 6
two times.
μ
