|Home > About this Database > 4.Experimental Procedures|
|Last Updated (September 15, 2003)|
About this Database
Malaria Full-Length cDNA project
I) Cultivation of parasites
Plasmodium falciparum parastite 3D7 strain was obtained from Prof. D. Walliker of Edinbough University and propagated as described by W Trager and JB Jensen (Science 193: 673-675).
Plasmodium yoelii 17X non-lethal strain was obtained from Prof. S. Waki and propagated in vivo in ICR mice. Blood was filtrated through Plasmodipur filter to remove contaminating white blood cells.
II) Isolation of RNA.
Cytoplasmic RNA and Poly A+ RNA were isolated using standard methods. Oligo-dT cellulose was obtained from Collaborative Biomedical Products and Roche.
Oligo-capping was performed as described (Maruyama and Sugano, 1994), with some modifications. In brief, 10 to 50 ug of polyA+ RNA was treated with 1.2 units of bacterial alkaline phosphatase (BAP; TaKaRa) in 100ul of 100mM Tris-HCl (pH 8.0), 5mM 2-mercaptoethanol with 100 units of RNasin (Promega) at 37oC for 60 min. After extraction with phenol:chloroform (1:1) and ethanol precipitation, the polyA+ RNA was treated with 20 units of tobacco acid pyrophosphatase (TAP) in 100ul of 50mM sodium acetate (pH 5.5), 1mM EDTA, 5mM 2-mercaptoethanol with 100 units of RNasin at 37oC for 60 min. After phenol:chloroform extraction and ethanol precipitation, 2 to 4ug of the BAP-TAP treated polyA+ RNA were ligated with 0.4ug of 5'-oligo (KM-02; 5'-AGC AUC GAG UCG GCC UUG UUG GCC UAC UGG-3') using 250 units of RNA ligase (TaKaRa) in 100ul of 50mM Tris-HCl (pH7.5), 5mM MgCl2, 5mM 2-mercaptoethanol, 0.5mM ATP, 25% PEG8000 with 100 units of RNasin at 20oC for 3 hours.
IV) cDNA synthesis.
After unligated 5'-oligo was removed, cDNA was synthesized with RNaseH free reverse-transcriptase (Superscript II, Gibco BRL). Ten picomoles of dT adapter-primer (5'-GCG GCT GAA GAC GGC CTA TGT GGC CTT TTT TTT TTT TTT TTT-3') was used in 50ul with 2 to 4ug of oligo-capped polyA+ RNA. The reaction conditions were as recommended by the supplier and reactions were incubated at 42oC for 3 hours.
V) cDNA amplification.
After first-strand cDNA synthesis, RNA was degraded in 15mM NaOH by incubating at 65oC for 1 hour. The cDNA which is made from 1ug of "Oligo-capped" polyA+ RNA was amplified in a volume of 100ul using an XL PCR kit (Perkin-Elmer) with 16pmol of 5' (5'-AGC ATC GAG TCG GCC TTG TTG-3') and 3' (5'-GCG GCT GAA GAC GGC CTA TGT-3') PCR primers. For dT-adapter primer primed cDNA, amplification were carried out by 15 cycles of PCR reactions at 94oC for 1 min, 58oC for 1 min, and 72oC for 10 min. PCR products were extracted with phenol:chloroform (1:1) once, ethanol precipitated and digested with SfiI. SfiI-digested PCR products were separated by agarose gel electrophoresis and products longer than 1 kb were isolated and cloned into DraIII-digested pME18S-FL3. In this way, we could clone the cDNA into the vector in an orientation-defined manner.
Plasmid DNA was isolated using PI-100 and PI-200 auto-plasmid-isolators (KURABO).
were determined by the dideoxy termination method using an AutoCycle sequencing
kit (Pharmacia) and a reaction robot R. O. B. DNA processor (Pharmacia)
or a BigDye sequencing kit (ABI). The sequence was read using an ALF DNA
(Pharmacia) and an ABI 377XL (ABI) auto-sequencer.
welcome your comments and feedback about our database.
Please feel free to contact us...... email@example.com