Biomedical Engineering Reference
In-Depth Information
10. 37 °C microbiological incubator (Function line, Haraeus/
Thermo Fisher Scientifi c; Catalog # 50042308) or similar.
11. 37 °C shaker-incubator (Multitron, Infors HT, Basel,
Switzerland) or similar.
12. 0.2 mL PCR tubes.
13. Ready-mix PCR master kit, 2× stock (Amplicon, Skoulunde,
Denemark; Catalog # 180301) or equivalent.
14. Temperature-controlled microcentrifuge (Eppendorf,
Hamburg, Germany; Catalog # 5417R) or equivalent.
15. Sterile toothpicks.
16. Glycerol (J.T. Baker, Phillipsburg, NJ, USA; Catalog #
2136-01) or equivalent.
3
Methods
Primer design is a critical step for success of the TPCR reaction.
Primers include at the 5
3.1
Primer Design
-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 amplifi cation of the gene [
12
]. The total primer length
should be 50-60 bases. The length of the overlapping sequence to
the destination vector can range from 20 to 40 bases with the
recommended length of 30 bases (
see
Fig.
2
). 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.
′
3.2 Cloning by
Transfer-PCR (TPCR)
For the TPCR reaction we use a two-stage amplifi cation process.
In the fi rst stage, 13 cycles are performed to amplify the target
DNA. In the second stage, additional 20 longer amplifi cation
cycles are used for incorporation of the PCR product into the
destination vector (
see
Note 4
). The most critical parameter for
effi cient assembly of DNA fragments using the TPCR reaction is
the fi nal primer concentration which should be in the range of
10-20 nM for both the forward and the reverse primers [
12
]. This
primer concentration is much lower than that being used for a
routine PCR amplifi cation, which is in the range of 0.4-0.8
M for
each primer. The example described in the protocol for cloning by
TPCR is transferring the gene encoding IFN
μ
α
8 from the donor
plasmid pPIC9K-IFN
α
8 into the expression vector pET28-TevH
(Figs.
2
and
3
).
1. Set up TPCR reactions in 0.2 mL tubes, in a fi nal volume of
50
μ
L, as follows (
see
Note 5
):
