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Simple method for cDNA amplification starting from small amount of total RNA.

We describe a novel simple PCR-based technique for construction of cDNA libraries starting from the small samples of cells or tissues. This technique is based on the insertion of inverted terminal repeats (ITR) into amplified cDNA which causes a part of molecules to generate "pan"-type structures at each cycle of PCR amplification. This allows to avoid generation of the primer dimmer and makes possible the regulation of an average length of amplified sequences varying in concentration of primers.

Animals↗
PubMed1976

Ribosomal RNA synthesis in the mycelium of Phycomyces blakesleeanus. High molecular weight RNA.

Total RNA was extracted from 24 h old mycelia of Phycomyces blakesleeanus which had been grown in liquid culture. Heavy molecular weight RNA was fractionated on 2.6% polyacrylamide gels. 1. The main cytoplasmic ribosomal RNA fractions had molecular weights of 1.34 and 0.72-16(6). 2. [3H]Uridine pulse labelling of cultures revealed an initial ribosomal RNA precursor of molecular weight 2.5-10(6). 3. An intermediate precursor for the 1.34 - 10(6) dalton peak was found, having a molecular weight of 1.6-10(6). 4. Pulse-chase experiments, using [3H]methionine, showed the flow of synthesis as being (see article). 5. Mature 0.72-10(6) dalton RNA appeared prior to the 1.34-10(6) dalton RNA but no immediate precursor for it was detected.

Electrophoresis, Polyacrylamide Gel↗
PubMed1981

Identification of Limulus polyphemus haemocyanin messenger RNA.

Total RNA was isolated from cyanoblast-containing tissue taken from behind the compound eye of the horseshoe crab, Limulus polyphemus. Poly(A)-containing RNA separated from this by affinity chromatography on oligo(dT)-cellulose was translated in the rabbit reticulocyte haemolysate system in the presence of L-[35S]methionine. By using an antiserum to Limulus haemocyanin, polypeptides were isolated from the translation products which had a similar mobility to the authentic Limulus haemocyanin polypeptides as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

Animals↗