Biomedical Engineering Reference
In-Depth Information
Fig.
1
(
a
) An example plasmid, pExample based on pET28a (Novagen), consisting of four PCR fragments (
grey
)
which when digested with T4 DNA polymerase and combined together create the 11,771 bp plasmid. The 3
′
end of each fragment matches the 5
end of the next fragment. Primer binding sites for each fragment are
indicated (forward,
dark green
; reverse,
light green
). Annotations: open reading frames (
yellow
), origins of
replication (
blue
), T7 terminator (
orange triangle
), T7 promoter (
purple triangle
). (
b
) Detailed example of the
overlap between Fragment 1 and Fragment 2. Primers (
light
/
dark green
) were designed such that a 38 bp
overlap (
orange
) is created between the two fragments after PCR and T4 DNA polymerase digest. This overlap
has a
T
m
of 77 °C and is stable during “ligation” at 37 °C. The forward primer binds the template with an initial
T
m
of 57 °C (initial primer site,
dark blue
), while the reverse primer has an initial
T
m
of 65 °C (initial primer site,
light blue
)
′
2.1 Sodium Borate
Gel Electrophoresis
1. 20× Sodium Borate (SB) buffer: 0.2 M NaOH, 0.73 M boric
acid, pH 8.0 [
13
]. Dissolve 8 g of NaOH in 500 mL of water
followed by 45 g of boric acid. Make up to 1 L with water
(
see
Note 1
).
2. 1× SB: 10 mM NaOH, 36.5 mM boric acid, pH 8.0. Mix
50 mL of 20× SB buffer with 950 mL of water. Store at room
temperature (
see
Note 1
).
3. 10× sample buffer: 0.25 % (w/v) bromophenol blue, 0.25 %
(w/v) xylene cyanol FF, 30 % (w/v) glycerol. Dissolve 0.05 g
bromophenol blue and 0.05 g xylene cyanol FF in 10 mL of
water. Add 6 mL of glycerol and make up to a fi nal volume of
20 mL with water, and store at 4 °C.
