A pyramidal-shaped fruit of Thaumatococcus daniellii kept at 80’C was powdered by mortar and
pestle in the presence of liquid nitrogen.
About 0.5 mg of the powder of the frozen tissue was dissolved in 0.4 mL of extraction buffer.
The supernatant was mixed with oligo (dT) cellulose suspension and centrifuged at 10,000 g for
10 s.
The precipitates were washed 5 times with 1 mL of high-salt buffer and then washed twice with
1 mL of low-salt buffer.
After washing, the supernatants were suspended in 0.3 mL of low-salt buffer and applied to the
MicroSpin column.
After centrifugation at 10,000 g for 5 s, the column was washed with 0.5 mL of low-salt buffer
three times and, finally, the mRNA was eluted with 0.4 mL of elution buffer that was preheated
to 65’C by centrifugation (10,000 g, 5 s).
First-strand cDNA synthesis was performed using a First-strand cDNA Synthesis Kit
(Pharmacia, Uppsala, Sweden) as follows.
Precipitated mRNA was dissolved in 20 AL DEPC water and heated at 65’C for 10 min.
After being chilled on ice, isolated mRNA was reverse-transcribed using 1 AL of pd (N)6
primers, 1 AL of DTT solution, and 11 AL of bulk First Strand cDNA Reaction Mix at 37jC for 1 h.
The thaumatin gene was amplified by PCR using the first-strand cDNA as a template, and
5V-GCCACCTTCGAGATCGTCAAC-3V and 5V-CCTAGGGGCAGTAGGGCAGAA-3V as
primers (underlined Avr II site).
The PCR reaction was conducted using Taq polymerase for 1 cycle of 94jC for 1 min,
and 30 cycles of 96’C for 30 s, 67’C for 30 s, and 72’C for 1 min, and then 1 cycle of 72’C for 10
min.
The PCR product was analyzed by 1.2% agarose gel electrophoresis.