John M. S. Bartlett.pdf - Bio-Nica.info

110 Wang and Young
Fig. 3. Expressed sequence tag of PG23. Sequence information of 277 bp was derived
from a cloned DNA fragment using the DD-PCR method (5). Double-strand sequences are shown
for easy identification of primer’s location (underlined). A 10-nucleotide primer (5′-GATCT-
GACTGC-3′) and an olio-dT primer (5′-TTTTTTTTTTTTCA-3′) were used in DD-PCR for
the original cloning of PG23. AGSP (5′- GGAACAGTCTGAGCTCTAAGCTAGG-3′) and
NAGSP (5′-TCTAGAAGGATAAGTTCACGAGGGCC-3′) were designed for the 5′-RACE of
full-length cDNA of PG23.
The sample was then subjected to 30 cycles of PCR on a thermocycler using the following
parameters: 94°C for 30 s, 60°C for 30 s, and 68°C for 3 min. For RACE PCR of GC rich
mRNAs, addition of some co-solvent may increase PCR efficiency (see Note 11).
7. Agarose gel electrophoresis of 5′-RACE PCR products: The above AGSP and NAGSP
were used in two independent RACE PCR. Then, 20 µL was loaded on a 1% agarose gel
and electrophoresed in 1× TAE buffer at 75 volts for 2 h. Each of these antisense primers
produced bands of expected size on ethidium bromide stained agarose gels when coupled
with the AP-1 primer (Fig. 1B, lanes 1 and 2). The band produced by the NAGSP (Fig. 1B,
lane 2) is slightly smaller compared with the one generated by AGSP (Fig. 1B, lane 1),
confirming that the bands are true RACE products. When only AGSP, NAGSP, or AP-1
were used in PCRs, these sharp bands disappeared (data not shown). The DNA bands of
proper size were excised from agarose gel with a clean, sharp scalpel. DNA was extracted
from the gel using the QIAEX II kit according to the manufacturer’s instruction. A 15-µL
elution was saved for later use.
8. The gel-purified DNA (5 µL) was used for cloning into the pGEM-T vector. Sequence
information for PG23 mRNAs of 1.4 kb and 2.0 kb was subsequently determined (data
not shown).
9. Nested second PCR: If the first PCR with AGSP and AP-1 is insufficient to produce a
visible band(s) of expected size, a second round of PCR with NAGSP and AP-2 (nested
within AP-1 primer) could be performed using 2.5 µL of 1:100 dilution of the first-PCR
product as template. This second round of PCR usually yields defined fragments of
expected size on ethidium bromide stained agarose gel. Sometimes no visible band