Biomedical Engineering Reference
In-Depth Information
7. Inoculate positive clones into 10 mL LB medium supplemented
with antibiotic in 50 mL conical tubes. Concentration of the
antibiotic depends on the type of the antibiotic used.
8. Grow bacteria with shaking at 37 °C, for 16-20 h.
9. Harvest cells and extract DNA using a plasmid DNA purifi ca-
tion kit (
see
Note 15
).
10. Measure DNA concentration using a NanoDrop spectrophotometer.
11. Send the 2-3 positive selected clones for DNA sequencing.
12. Transform plasmids from each positive clone to competent
BL21(DE3)
E. coli
(
see
Note 16
) cells and proceed with pro-
tein expression experiments as described previously [
13
,
14
].
4
Notes
1. Primers (forward and reverse) for the RF reaction include at the
5
-end a vector-specifi c sequence, complementary to the site of
integration into the recipient vector, and at the 3
′
-end a
sequence complementary to the gene of interest used for ampli-
fi cation of the gene [
9
,
11
] (
see
Table
1
for primer sequences).
The total primer length is 50-60 nt. The length of the overlap-
ping sequence to the destination vector can range from 20 to
40 nt with the recommended length of 30 nt. The length of the
gene-specifi c sequence is variable and should be designed to
satisfy the melting temperature (
Tm
) requirements. The
Tm
for
the gene-specifi c sequence should be between 60 and 70 °C,
where A or T, and G or C, each contributes to the
Tm
2 and
4 °C, respectively. Synthetic primers were ordered from
Integrated DNA Technologies (IDT; Leuven, Belgium) or
Sigma-Genosys (Rehovot, Israel). Primers up to 60 nt are
ordered with only basic desalting purifi cation. Longer primers
are purifi ed either by HPLC or by SDS-PAGE.
′
2. We found that preparation of competent cells according to the
procedure previously described [
15
] is best suited for the high
effi ciency of the RF reaction. In this procedure, cells are grown
at 18 °C prior to harvesting and preparation of the competent
cells. 90-100
μ
L of competent cells are used for transforma-
L of DpnI-treated RF reaction.
3. On a routine basis we use plasmid DNA obtained from various
sources as the template for amplifi cation of the gene of inter-
est. However, we have also used other sources for amplifi cation
such as chromosomal DNA from various microorganisms and
cDNA libraries. When using DNA sources other than plasmid,
optimization of the PCR amplifi cation might be essential
(
see
Note 4
).
tion of 1-10
μ
