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

108 Wang and Young
Fig. 1. (A) Northern blot analysis: 10 µg of total RNA were loaded in each lane; Pineal
(lane 1), brain (lane 2), lung (lane 3), and kidney (lane 4). (B) RACE PCR: full-length
cDNAs (1.4 kb and 2.0 kb) were obtained using a long template PCR-based RACE technique.
Two primers, AGSP (5′- GGAACAGTCTGAGCTCTAAGCTAGG-3′, lane 1) and NAGSP
(5′-CTAGAAGGATAAGTTCACGAGGGCC-3′, lane 2), were designed using a 277-bp
sequence information from a sequenced DD-PCR product (Fig. 2) and coupled with AP-1
for 5′-RACE PCR.
dNTP, 1 µL of 10 µM oligo-dT primer (5′-CACTATAGGCCATCGAGGCC(T)20MN-3′),
and 1 µL of SuperScript II (RNase H-) reverse transcriptase (added last; see below). The
reaction was performed in a thermocycler with the following parameters: 70°C for 10 min,
42°C for 50 min, and 50°C for 15 min. SuperScript II RNase H- reverse transcriptase
(1 µL) was added to the reaction mixture after a 5-min incubation at 42°C. The reaction
mixture was placed on ice before the next step.
2. Second-strand cDNA synthesis: The following components for second-strand cDNA
synthesis were added to the above 10-µL first-strand reaction mixture: 48.4 µL of highquality
H 2 O, 16 µL of 5× second-strand buffer, 1.6 µL of 10 mM dNTP mixture, and 4 µL
of 20× second-strand enzyme cocktail. After mixing the contents briefly with gentle pipetting,
the reaction was placed in the thermocycler for incubation at 16°C for 1.5 h. Then, 2 µL of
T4 DNA polymerase was added and mixed well, and incubation continued at 16°C for
another 45 min to create blunt end. Finally, 4 µL of the EDTA/Glycogen mixture (glycogen
helps bring down the cDNA in a later precipitation) was added to stop the reaction.
3. Purification of blunt-ended double-stranded cDNA: For efficient recovery of the relatively
small amount of the cDNA, the general phenol/chloroform extraction was performed twice
using 100 µL of phenolchloroformisoamyl alcohol (25241). The supernatant plus
0.5 volume of 4 M ammonium acetate and 2.5 volumes of 100% ethanol were placed at –20°C
for at least 1 h and centrifuged for 10 min at 4°C. The recovered DNA pellet was washed in
1 mL of 75% ethanol and resuspended in 10-µL high-quality water after air-drying.
4. Adaptor ligation: A partially double-stranded adaptor was ligated to both ends of the
above-purified double-stranded cDNA. Ten microliters of ligation mixture containing
5 µL of purified blunt-ended double-stranded cDNA, 2 µL of adaptor, 2 µL of 5× ligation