Biomedical Engineering Reference
In-Depth Information
localization of a Bax mutant (Fig.
3b
,
see
Note 9
), indicating
that in-frame fusion occurred effi ciently by the used overlap
sequences.
3.2 Minimum
Annealing Sequences
for Primer Design
When Used with an
Additional Sequence
When a primer contains an additional sequence, only 9-12 nucleo-
tides long annealing sequence can be used for PCR without any
special attention [
9
]. As the minimum GC-rich annealing sequences,
we propose 9 nucleotides long 5CG9 (5
′
CCCCCGGGG3
′
), 9C
(5
) for design-
ing primers when it is used with additional sequences. For the prep-
aration of templates containing GC-rich annealing sequences, fi rst
PCR is performed with primers containing these GC-rich sequences.
Then, the templates are used for the second PCR with the primers
containing the minimum GC-rich annealing sequences and desired
additional sequences.
′
CCCCCCCCC3
′
), and 3CG9 (5
′
CCCGGGCCC3
′
1. Use the following forward primers; 5CG12-yEGFP2+4,
5CG9-yEGFP2+4, 5CG6-yEGFP2+4, 12C-yEGFP2+4,
9C-yEGFP2+4, 6C-yEGFP2+4, 3CG12-yEGFP2+4, 3CG9-
yEGFP2+4, and 3CG6-yEGFP2+4 (
see
Note 2
for sequence
nomenclature) with a reverse primer yEGFP2+720c to prepare
yEGFP2 (yeast-codon-optimized EGFP) templates by PCR.
2. Mix 6.6
μ
L of sterile water, 1
μ
L of 10× KOD Plus buffer, 1
μ
L
of 2 mM dNTPs, 0.4
μ
L of 25 mM MgSO
4
, 0.2
μ
L of 10
μ
M
forward primer, 0.2
μ
L of 10
μ
M reverse primer yEGFP+720c,
0.4
L of
K
.
marxianus
total DNA (RAK8961) containing
pKM141 plasmid (0.5 ng/
μ
μ
L) (
see
Note 10
), and 0.2
μ
L of
L.
3. Run a PCR program comprising an initial denaturation step at
94 °C for 1 min, followed by 30 cycles of 94 °C for 20 s, 60 °C
for 30 s, and 68 °C for 2 min.
4. Check the PCR products by agarose gel electrophoresis. Each
PCR product should yield single bands (0.7 kb) upon gel
analysis.
5. Adjust the DNA concentration to 0.5 ng/
KOD Plus polymerase. Total volume: 10
μ
L. These PCR
products contain GC-rich sequences at the ends of the forward
primer side.
6. Perform second PCR with the following forward primers;
NcSu9(67-99)-5CG12, NcSu9(67-99)-5CG9, NcSu9(67-
99)-5CG6, NcSu9(67-99)-12C, NcSu9(67-99)-9C,
NcSu9(67-99)-6C, NcSu9(67-99)-3CG12, NcSu9(67-99)-
3CG9, and NcSu9(67-99)-3CG6 with a reverse primer
yEGFP2+720c, respectively (
see
Note 11
).
7. Mix 6.6
μ
μ
L of sterile water, 1
μ
L of 10× KOD Plus buffer, 1
μ
L
of 2 mM dNTPs, 0.4
μ
L of 25 mM MgSO
4
, 0.2
μ
L of 10
μ
M
forward primer, 0.2
μ
L of 10
μ
M yEGFP2+720c primer,
0.4
μ
L of template DNA (0.5 ng/
μ
L), and 0.2
μ
L of KOD
Plus polymerase. Total volume: 10
μ
L.
