Biomedical Engineering Reference
In-Depth Information
4. The components and the reaction conditions for the PCR
amplifi cation can be modifi ed if needed. Addition of dimethyl
sulfoxide (DMSO), 5-10 %
v
/
v
, might be essential for success-
ful reaction if the target DNA has high GC content. Performing
a gradient PCR or changing the annealing temperature might
be essential, in certain cases, to obtain successful amplifi cation.
If large DNA fragments are amplifi ed increasing the elonga-
tion time might be essential.
5. Analyzing the PCR amplifi cation reactions on agarose gel is
essential step to ensure that the correct size PCR product was
obtained as the major band. If no PCR product was obtained,
reexamine the primer design. In addition, make sure that
source DNA used is correct and if needed alter the PCR condi-
tions (
see
Note 4
).
6. We highly recommend purifying the PCR product(s) directly
from the PCR mix and not from the agarose gel. We have
noticed that purifying the PCR product following extraction
from the agarose gel resulted in loss of much of the DNA and
reduced yield of gene integration into the destination vector in
subsequent steps. Removal of the PCR product from an aga-
rose gel is an optional step when multiple bands are obtained
in the initial PCR amplifi cation step.
7. In certain cases altering the components for the integration
step might be essential. If addition of dimethyl sulfoxide
(DMSO) was essential for PCR amplifi cation in the fi rst stage
(
see
Note 4
), it should be used in this stage, as well. On a rou-
tine basis, we used 20 ng from the destination vector and
100 ng from each PCR product (mega-primers). However,
increasing the amount of the mega-primers (up to 250-300 ng
per reaction) might improve, in certain cases, the effi ciency of
integration. If more than two PCR products (as specifi ed in
the protocol) are used for simultaneous integration into the
destination vector use the same DNA concentration for the
additional mega-primers.
8. If the total size of the newly synthesized vector (DNA insert(s)
plus destination vector) is large (>15 kb) increasing the elon-
gation time might be essential. The recommended time for the
elongation step for the Phusion DNA polymerase is 15-30 s/
kb. In addition, optimization of the integration reaction using
gradient PCR might be essential, primarily when multiple
mega-primers with various length and complexity are used
simultaneously.
9. Analyzing the RF reaction on agarose gel is an optional, but
highly recommended, step. The analysis should give a strong
indication on how successful the assembly reaction worked.
If high molecular band, corresponding to the newly synthe-
