Biomedical Engineering Reference
In-Depth Information
Fig.
6
In-frame addition of a peroxisome-targeting sequence to RFP/GFP through GC-rich minimum annealing
sequences. (
a
)
URA3
-
TDH3
promoter-yEmRFP template was amplifi ed with primers containing indicated mini-
mum annealing sequences (
bold
). The amplifi ed products were used as templates for the second PCR with
primers containing minimum annealing sequences (5CG9, 9C, 3CG9, and 3GC9) and a complementary
sequence (
italics
) of SKL for peroxisome targeting. The PCR products were used for transformation to the yeast
Kluyveromyces marxianus ura3
mutant strain. Transformants showed punctate distribution of RFP signals.
Number of the correct in-frame addition of the SKL sequence was counted and compared to that through the
RFP C-terminal sequence. (
b
) Second PCR amplifi cation for the addition of SKL sequence. (
c
) Cellular localiza-
tion of yEmRFP and EGFP with SKL attached by 3CG9 annealing was shown.
FL
fl uorescence,
BF
bright fi eld
These PCR products are used as templates for the addition of SKL
sequence to the RFP C-terminus (Fig.
6
).
1. For the preparation of templates, use a forward primer
ScTDH3+1000 with the following reverse primers: 3CG9-
yEmRFP+687c, 5CG9-yEmRFP+687c, and 9C-yEmRFP+687c,
to prepare templates of yEmRFP by PCR (
see
Note 2
for
sequence name).
2. Mix 5.4
μ
L of sterile water, 2
μ
L of 5× PrimeSTAR GXL buf-
fer, 0.8
μ
L of 2.5 mM dNTPs, 0.3
μ
L of 10
μ
M forward
primer, 0.3
L of
K
.
marxianus
total DNA (RAK9172) containing pKM382 plasmid (0.5 ng/
μ
μ
L of 10
μ
M reverse primer, 1
μ
L), and 0.2
μ
L of GXL DNA polymerase. Total volume:
L (
see
Note 10
).
3. Run a PCR program comprising an initial denaturation step at
98 °C for 4 min, followed by 30 cycles of 98 °C for 10 s, 55 °C
for 15 s, and 68 °C for 5 min.
4. Check the PCR products by agarose gel electrophoresis. Each
product showed a clear DNA band (5.3 kb, data not shown).
5. Adjust the DNA concentration to 0.5 ng/
10
μ
L (
see
Note 14
).
These PCR products contain GC-rich sequences at the ends of
the reverse primer side.
μ
