Biomedical Engineering Reference
In-Depth Information
5. Adjust to 0.1-0.5 ng/
L. If the DNA concentration cannot be
measured, approximately 100 times dilution of the PCR prod-
uct will give this range.
6. Concentration of the DNA fragments is critical for fusion
PCR. If higher concentration of DNA fragment is used, unex-
pected bands or smear appears but if it is lower or unequal
between two fragments, low or no yield of fusion DNA is
obtained [
3
].
7. Fusion PCR requires relatively higher annealing temperatures.
We routinely use 60 °C. Lower annealing temperatures will
not give better results.
8. Sometimes low PCR amplifi cation from certain templates was
experienced, possibly due to strong GC annealing to unex-
pected region of the template. In such cases, 10CA/10TG and
10CT/10AG overlaps [
17
] can be applied.
9. The used Bax mutant localizes at mitochondria in mammalian
cells without apoptotic signal. 5CGC and 3CGC showed simi-
lar results but the fusion construct via an EGFP C-terminal
sequence showed cytoplasmic localization in addition to mito-
chondrial localization. This may be caused by unexpected
products shown as smear in Fig.
3a
. 5CGC and 3CGC encode
PPGAP and PGPGP, respectively, and these linker sequences
did not affect Bax localization [
3
].
10. A replicating plasmid is constructed and maintained in
K. marxianus
. The plasmid region can be amplifi ed directly
from yeast total DNA (chromosomal DNA) as a template.
11. NcSu9(67-99) is a 33-nucleotide long sequence of a mito-
chondrial targeting signal of
Neurospora crassa
ATPase sub-
unit 9 [
10
] and used as an additional sequence of the primers
in this case.
12. Higher annealing temperatures than 60 °C gave lower yield.
13. We routinely use 3CG9 and 9C, and these can be used simul-
taneously. In yeast gene manipulation, gene disruption is per-
formed by transformation of a selection marker gene
containing homologous sequences, usually 40-60 nucleotides
in length, at the both ends. We use 9C and 3CG9 minimum
annealing sequences for the addition of the homologous
sequences. If another marker gene was required for the dis-
ruption, the same primers of the homologous sequences can
be used for the amplifi cation of the other markers that attached
9C and 3CG9 at the ends. Genome-wide gene manipulations
often use thousands of DNA constructs. Even if only 5-10
nucleotides were reduced in primer design, it will greatly save
primer cost for genome-wide design [
18
], and the same set of
primers is possibly used for the other projects.
μ
