lithium-chloride and isopentyl-alcohol

A quick, simple and reliable method of extracting DNA from sweetpotato (Ipomoea batatas (L.) Lam.) has been developed. The method was applied successfully for extraction of total DNA from leaves and total RNA from leaves and various tissues. The yield of DNA extracted by this procedure was high (about 1 mg/g leaf tissue). The extracted DNA was completely digested by restriction endonucleases indicating the absence of common contaminating compounds. The absorbancy ratios of A260/A230 and A260/A280 of isolated RNA were approx. 2 and the yield was about 0.2 mg/g fresh wt. CIPK and tublin genes were successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. The total DNA and RNA isolated by this method was of sufficient quality for subsequent molecular analysis.

Topics: 2-Propanol; Blotting, Southern; Chloroform; DNA, Plant; Electrophoresis, Agar Gel; Ipomoea batatas; Lithium Chloride; Pentanols; Plant Components, Aerial; Plant Leaves; Plant Roots; Protein Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Plant; RNA, Ribosomal; Spectrophotometry, Ultraviolet; Tubulin

Isolation of full-length mRNA without degradation is critical in the study of in vivo gene regulation and transcription, cDNA synthesis and reverse transcription (RT)-PCR. It is particularly difficult to isolate full-length mRNA from thermophiles, which have higher turnover rates of mRNA degradation. Mastigocladus laminosus is a thermophilic heterocystous cyanobacterium. The assay of M. laminosus cell lysates showed that RNase activity was high and was resistant to the conventional guanidine thiocyanate and 2-mercaptoethanol denaturation methods. The mRNA isolated by several conventional methods was completely degraded. A method was developed to purify full-length mRNA by a combination of fast cooling, vanadyl-ribonucleoside-complex inhibition, phenol-chloroform-isoamyl alcohol extraction, lithium chloride precipitation and the lysing of cells with the French Press. This method produced high-quality, full-length mRNA in high yield. Purified mRNA was suitable for Northern blotting, cDNA synthesis and RT-PCR. This method could be applicable to other thermophiles in which the RNase activity is high and/or is resistant to guanidine thiocyanate.

Topics: Blotting, Northern; Chemical Precipitation; Chloroform; Cold Temperature; Cyanobacteria; Electrophoresis, Agar Gel; Escherichia coli; Guanidines; Lithium Chloride; Mercaptoethanol; Nucleic Acid Denaturation; Pentanols; Phenol; Ribonucleases; Ribonucleosides; RNA, Bacterial; RNA, Messenger; Thiocyanates; Vanadium Compounds