Biology Reference
In-Depth Information
standard cell culture conditions until most cells show cytopathic
effect (CPE) (
see
Note 23
).
20. Repeat
steps 12-17
.
21. Centrifuge sample for 10 min at 3,500 ×
g
at room temperature
and transfer the new lysate into a fresh Eppendorf tube. Store
the fi nal lysate at −80 °C or directly continue with Ad amplifi -
cation according to standard protocols.
4
Notes
1. To propagate electrocompetent
E. coli
SW102, plate out fro-
zen stocks of SW102 cells on LB plates and incubate them at
32 °C overnight and carry out the
steps 5-11
of Subheading
3.3
using LB without antibiotics for all cultures. Electroporate
competent cells with 10 ng of purifi ed Ad-BAC using 2,500 V,
200 W, and 25
μ
F in Gene Pulser (or equivalent electropora-
tion equipment).
2. Ad-BACs are very stable and can be easily retransformed with
high fi delity. Therefore a simple restriction analysis is suffi cient
to ensure their integrity in a new
E. coli
strain after electro-
transformation. However, we do not recommend using any
other method for transformation of BACs.
3. The pgalK-Kn construct and its nucleotide sequence are avail-
able from the corresponding author upon request.
4. We recommend the Expand High Fidelity PCR System from
Roche. In this case, mix the template with the 1
μ
L of each
primers (from a 100
L
10× PCR reaction buffer, 2.5 U polymerase, add deionized
PCR quality water to fi nal volume of 100
μ
M stock), 2
μ
L 10 mM dNTPs, 10
μ
L. Run the PCR for
35 cycles; in the fi rst 18 cycles touch-down the annealing tem-
perature from 62 to 45 °C and set the annealing temperature
at 45 °C for additional 17 cycles. Allow 2 min for annealing
time. Elongation in all 35 cycles should be set at 68 °C for
2 min, and denaturation in all 35 cycles should be at 94 °C for
30 s.
5. Residual PCR template is the main source of false-positive
background colonies during the fi rst targeting. Therefore, a
complete
Dpn
I digestion of template plasmid after PCR is
essential. Since the PCR-derived DNA is not methylated, the
residual plasmid-derived template DNA can be removed by
Dpn
I digestion, which only cuts
dam
methylated DNA. To
make sure that the template pgalK-Kn is well methylated, it
should be propagated in
dam
-positive
E. coli
strain (DH10B).
μ
