Potato virus X isolation and purification and coat protein gene cloning
In this work we describe the construction of potato virus X coat protein (PVX-CP) gene clones from a Colombian isolate. Virus was isolated from field potato plants cultured at páramo de San Jorge. PVX particles were purified from Nicotiana tabacum leaves infected with a local lesion taken from Datura stramonium plants. The genomic RNA present in the virus particles consisted of a single species of about 6 Kb. cDNA synthesis representing genomic RNA was followed by Digoxigenin incorporation after priming with oligonucleotide 4DT/KPN. PCR amplification of CP gene sequence was made by using oligonucleotides OX6 and L/Kpn. An expected fragment of 870 bp, besides some 600-123 bp fragments, was obtained after PCR. The blunt-ended fragments were clonned into the PCR cloning pMOSblue plasmid. The insert size of the selected recombinant cDNA clones was determined by restriction analysis of the plasmidDNAwith Sma I and Hind III enzymes. Positive clones were also screened by direct colony PCR screening. The selected recombinant cDNA clones were stored in glycerol at .70 °C.
| Main Authors: | , , , |
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| Format: | Digital revista |
| Language: | spa |
| Published: |
Universidad Nacional de Colombia - Sede Bogotá - Instituto de Biotecnología
2001
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| Online Access: | https://revistas.unal.edu.co/index.php/biotecnologia/article/view/30063 |
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