Biomedical Engineering Reference
In-Depth Information
which contains the appropriate
attB
recombination signal and
12-16 nt of overlapping sequence with the fi rst primer. A mix-
ture of PCR products will be produced, but only the full length
product will have the
attB
recombination sites necessary for
recombination to occur.
5. Adapter PCR can be used on both ends simultaneously by add-
ing two different adapter primers. One can also nest multiple
levels of adapter PCR to insert long 5
sequences if neces-
sary. Often, the same adapter primers can be used for any gene
which has a particular 5
′
or 3
′
sequence, thus minimizing the cost
and number of oligos which need to be generated for library
construction.
6. In cases where multiple large genes are to be combined (e.g., a
fusion of a protein of interest with a second protein of interest),
or when large deletions are desired, overlap PCR can be used
[
8
]. In this process, two separate PCR amplifi cations are carried
out with 20-25 bp of overlapping sequence between the 3
′
′
end
of gene 1 and the 5
end of gene 2. A third PCR is then carried
out using the fi rst two PCR products as templates along with
fl anking primers containing the
attB
sites. Again, the presence
of the
attB
sites during only the last round of PCR ensures that
no other side-products will be able to be cloned.
′
7. In addition to oligonucleotides for amplifi cation, oligonucle-
otides for sequence verifi cation of Entry clones are also
required. We generally order 1 primer for every 600 bp of
sequence, and an additional primer on the reverse strand that
is able to sequence back through the start of the gene. Typical
primer lengths are 22-24 nt, and they can be selected manually
or with the assistance of many common molecular biology
computer programs. Standard Gateway sequencing primers
can also be used to sequence into the gene of interest from
both directions in the Entry clone.
3.2 PCR
Amplifi cation
1. To a 200
μ
L thin-walled PCR tube, add 1
μ
L of each 5
μ
M
oligonucleotide primer, 0.75
L DMSO (
see
Note 2
),
50-100 ng template DNA (
see
Note 3
), and water to 12.5
μ
μ
L
fi nal volume.
2. Add 12.5
L 2× Phusion Master Mix HF, mix well, and carry
out the PCR amplifi cation using the following parameters: ini-
tial denaturation at 98 °C for 30 s, 20 cycles of (98 °C for 10 s,
55 °C for 30 s, and 72 °C for 30 s per kb of the expected prod-
uct), followed by a 10 min fi nal elongation at 72 °C, and cool-
ing to 4 °C (
see
Note 4
).
3. For adapter PCR (
see
Subheading
3.1
,
step 4
), after fi ve cycles
of amplifi cation, pause the thermal cycler, and add 1
μ
μ
L of
5
M adapter primer(s) to the tubes. Continue cycling for
additional 20 cycles.
μ
