Biomedical Engineering Reference
In-Depth Information
ATTC will preclude the choice of the sequence GAAT for any of
the other sites. Another requirement is to avoid the 16 palindromic
sequences (for four nucleotide fusion sites), since palindromic DNA
ends are compatible with themselves in the other orientation. For
enzymes that cleave on a four nucleotide sequence, 240 possible
sequences are therefore available.
3.3 PCR
Amplifi cation
of the Modules
1. A PCR mix is set up following the manufacturer's instructions.
For example, using KOD polymerase (
see
Note 2
), the following
conditions are used: 1
μ
L plasmid DNA (5-20 ng/
μ
L), 5
μ
L
of 10× buffer, 3
μ
L of 25 mM MgSO
4
, 5
μ
L of 2 mM dNTPs,
1.5
μ
L each of 10
μ
M sense and antisense primers, and 1
μ
L of
KOD Hot Start DNA polymerase (10 U/
μ
L, fi nal concentra-
L.
2. PCR is performed using the following cycling conditions:
(1) incubation at 95 °C for 2 min for polymerase activation,
(2) denaturation at 95 °C for 20 s, (3) annealing at 58 °C for
10 s the temperature for the annealing step can be adjusted for
specifi c primers, but the temperature of 58 °C usually works
well for most primers, (4) extension at 70 °C, the duration
depends on the length of the expected fragment (from 10 s/kb
tion 0.02 U/
μ
L) in a total reaction volume of 50
μ
for fragments smaller than 500 bp up to 25 s/kb for fragments
larger than 3 kb, see manufacturer's instructions);
steps 2
-
4
are repeated 34 times and are followed by a fi nal extension step
at 70 °C for 20 s-2 min (depending on fragment length). The
reaction is then kept at 12 °C until taken out of the
thermal cycler.
3. Of the PCR product obtained, 2
L is analyzed by gel electro-
phoresis to make sure that a product of the correct size has
been amplifi ed.
4. The amplifi ed fragment is purifi ed from remaining primers,
potential primer dimers, and remaining polymerase enzyme by
using the NucleoSpin
®
Extract II kit following the kit proto-
col. DNA is eluted from the column with 30-50
μ
L of elution
buffer (5 mM Tris-HCl, pH 8.5). In case several bands were
amplifi ed rather than only the expected fragment, the same
kit can also be used to cut and extract the appropriate DNA
fragment from an agarose gel.
μ
Cloning of the modules before assembly is optional since PCR
fragments can be assembled directly in a level 0 cloning vector
(
see
Note 3
). However, as mentioned above, cloning of level −1
fragments may be preferable to facilitate cloning of large level 0
modules. Cloning fragments before assembly may also be useful
if one wants to assemble several sequence fragment variants
combinatorially.
3.4 Blunt-End
Cloning of the
Modules
