Mattheus Nathalie, Ekramoddoullah Abul K M, Lee Stephen P
Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada.
Phytochem Anal. 2003 Jul-Aug;14(4):209-15. doi: 10.1002/pca.701.
Isolation of PinmIII cDNA homologues from white spruce tissues required a rigorous RNA extraction protocol developed following assessment of three previously reported conifer RNA extraction protocols. Total RNA was extracted via several purification steps designed to minimize binding of phenolics to nucleic acids and was then subjected to caesium chloride ultra-centrifugation. This procedure produced consistently high-quality, intact RNA from both needles and roots with spectrophotometric ratios of approximately 2.0 for both 260/280 nm and 260/230 nm. Total RNA was obtained from the roots of cold-hardened white spruce seedlings for cDNA library construction. More than 2 million recombinant phage particles were generated from 5 microg of a poly(A)+RNA fraction, and ca. 1.3 million cDNA particles were amplified for storage. Approximately 500,000 primary recombinant clones were screened with an heterologous PinmIII cDNA sequence yielding a unique clone, picgl, that was very similar to members of the PR10 gene family.
从白云杉组织中分离PinmIII cDNA同源物需要一个严格的RNA提取方案，该方案是在评估了之前报道的三种针叶树RNA提取方案后制定的。通过几个旨在尽量减少酚类物质与核酸结合的纯化步骤提取总RNA，然后进行氯化铯超速离心。该程序从针叶和根中均能持续产生高质量的完整RNA，260/280 nm和260/230 nm的分光光度比约为2.0。从抗寒白云杉幼苗的根中获取总RNA用于构建cDNA文库。从5微克的聚腺苷酸加尾RNA组分中产生了超过200万个重组噬菌体颗粒，并扩增了约130万个cDNA颗粒用于储存。用异源PinmIII cDNA序列筛选了大约50万个初级重组克隆，得到了一个独特的克隆picgl，它与PR10基因家族的成员非常相似。
