Rapid isolation of total RNA from mammalian tissues

A rapid procedure for the isolation of total RNA from small amounts of mammalian tissue (35 to 150 mg) is described. Tissues were homogenized in the presence of RNase inhibitors but in the absence of strong detergents. Contaminants were removed by phenol/chloroform extraction and Sephadex column chromatography. Total RNAs were precipitated with ethanol and sodium acetate. The RNAs isolated were intact and suitable for mRNA quantitation via Northern blot or slot-blot analyses. This procedure isolates total RNAs in high yield and purity, without CsCl ultracentrifugation, and is especially useful when mRNAs must be quantitated from many samples.     

Simultaneous and rapid isolation of bacterial and eukaryotic DNA and RNA: a new approach for isolating DNA.

A very simple procedure for the simultaneous preparation of genomic DNA and total RNA is described. The procedure is essentially the same for eukaryotes and prokaryotes except for the lysis buffer and can be used for small or large numbers of cells. Mammalian cells are lysed in sodium dodecyl sulfate and bacterial cells are lysed in Triton X-100, both in the presence of EDTA. RNA is obtained in the aqueous phase after phenol (acidic pH):chloroform:isoamyl alcohol extraction. DNA is eluted out of the organic phase (and the interface) into the aqueous phase by increasing the pH with highly basic 1 M Tris solution. The method is extremely rapid for small or large numbers of cells, and several large samples can be processed in one day. The qualities of both nucleic acids are excellent and the yield is high.     

Bacterial RNA isolation with one hour centrifugation in a table-top ultracentrifuge.

A procedure for the rapid preparation of cesium-chloride purified RNA from E. coli and the cyanobacterium Synechococcus sp. PCC7942 is described. Cells are lysed in modified sucrose, Triton X-100, EDTA, Tris buffer with phenol/chloroform. The cleared lysate is extracted further with phenol/chloroform and RNA is peleted by centrifugation through a 5.7 M CsCl cushion. High quality RNA can be prepared in three hours using this procedure.     

Extremely Rapid Extraction of DNA from Bacteria and Yeasts

A very simple and rapid method for extracting genomic DNA from Gram-negative bacteria, Gram-positive bacteria and yeasts is presented. In this method, bacteria or yeasts are lysed directly by phenol and the supernatant is extracted with chloroform to remove traces of phenol. The supernatant contains DNA that is suitable for molecular analyses, such as PCR, restriction enzyme digestion and genomic library construction. This method is reproducible and simple for the routine DNA extraction from bacteria and yeasts.     

Rapid purification of covalently closed circular DNAs of bacterial plasmids and animal tumor viruses

A rapid and simple purification of covalently closed circular (supercoiled) DNA from both bacterial clones (plasmids) and African green monkey cells (SV40) is presented. The method involves immediate treatment of lysed cells with sodium hydroxide, followed by neutralization and phenol extraction in high salt. After the extraction mixture is centrifuged, supercoiled DNA is found in the aqueous phase, the noncovalently closed DNA molecules form a white precipitate at the interphase, and proteins pellet. Contaminating RNA is eliminated from the aqueous phase by RNAse treatment and precipitation of the supercoiled DNA with polyethylene glycol. Residual polyethylene glycol is removed from the resuspended DNA by chloroform extraction. The purified supercoiled DNA is compatible with restriction enzymes, and is efficient at transforming both _χ1776 and HB101 bacterial hosts. Centrifugation in ethidium bromide-cesium chloride or sucrose gradients is not necessary. The method is virtually independent of the molecular size and gives good yields of supercoiled DNA. The technique is applicable to large-scale preparations and as a rapid “screening” procedure in which 20 to 30 samples can be easily purified within 5 to 6 h.     

A DNA extraction method for RAPD analysis from plants rich in soluble polysaccharides

A simple procedure for the isolation of DNA from mature leaf tissue was developed. This procedure purified DNA using Sephacryl S-1000 column and PEG 8000 precipitation. Polysaccharide-like components were successfully removed from DNA samples from species in which polysaccharides were found to be difficult to remove by phenol/chloroform extraction. The DNA obtained by this method was suitable for PCR, RAPD, enzyme digestion, and Southern-blot analyses.     

Plasmid purification by phenol extraction from guanidinium thiocyanate solution: development of an automated protocol.

We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid.     

A convenient procedure for the isolation of intact translatable mRNA by potassium iodide gradient centrifugation

High concentrations of KI were found to efficiently protect RNA against degradation by RNases. When a sufficient amount of solid KI was added to cell lysates or subcellular fractions (9 g per 10 ml), the solutions could be stored at room temperature for several days without measurable degradation of mRNA. Ribonucleic acids were selectively sedimented when these KI-containing solutions were centrifuged at 72,000 x g for 24 h. The RNA pellets were found to be readily dissolved in bidistilled water and the redissolved RNA could be immediately submitted to oligo(dT)-cellulose chromatography to isolate the poly(A)-containing RNA. However, extraction with phenol/chloroform was found to be necessary, if total RNA or poly(A)-minus RNA was to be analysed. This procedure was found to be superior to other methods currently in use - especially with respect to the isolation of intact, translatable high-molecular-weight mRNA.     

Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef

AIM: To develop an improved, rapid and sensitive sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef. METHODS AND RESULTS: Fresh ground beef samples were experimentally inoculated with varying concentrations of E. coli O157:H7. PCR inhibitors were removed and bacterial cells were concentrated by filtration and centrifugation, and lysed using enzymatic digestion and successive freeze/thaw cycles. DNA was purified and concentrated via phenol/chloroform extraction and the Shiga toxin 1 gene (stx1) was amplified using PCR to evaluate the sample preparation method. Without prior enrichment of cells in broth media, the detection limit was 103 CFU g-1 beef. When a 6 h enrichment step was incorporated, the detection limit was 1 CFU g-1 beef. The total time required from beginning to end of the procedure was 12 h. CONCLUSIONS: The sample preparation method developed here enabled substantially improved sensitivity in the PCR-based detection of E. coli O157:H7 in ground beef, as compared to previous reports. SIGNIFICANCE AND IMPACT OF THE STUDY: Superb sensitivity, coupled with quick turn-around time, relative ease of use and cost-effectiveness, makes this a useful method for detecting E. coli O157:H7 in ground beef.     

A simple, efficient method for the separate isolation of RNA and DNA from the same cells.

A method for the isolation of total cytoplasmic RNA and high molecular weight DNA from the same cells is described. Cells are gently lysed with NP40 in the presence of vanadyl ribonucleoside complex and the nuclei pelleted by centrifugation. RNA is purified by phenol/CHCl3 extraction of the lysate supernatant followed by ethanol precipitation. Protein is removed from high molecular weight DNA by salt-precipitation after nuclei are digested with proteinase K in the presence of sodium dodecyl sulfate. High yields of clean, intact RNA and DNA are obtained. A major advantage of the method is that it can be scaled down to quantitatively extract RNA and DNA from as little as 1000 cells.     

Rapid extraction of high molecular weight RNA from cultured cells and granulocytes for Northern analysis.

The study of messenger RNA in mammalian cells by Northern analysis requires the extraction of intact RNA in pure form. Although a number of reliable techniques have been developed for the purpose, most are fairly complex, involving steps such as ultracentrifugation and multiple extractions with large volumes of phenol and chloroform. When the number of cell samples to be analyzed is large, these techniques can be unwieldy. I now describe an RNA purification procedure which is simple enough to allow handling of a large number of cultured cell samples. It uses safe and inexpensive reagents and produces a high yield of pure total cell RNA, essentially free of DNA and ribonuclease, suitable for Northern analysis. The procedure also allows extraction of intact RNA from human granulocytes, cells which are rich in ribonuclease and contain very low amounts of RNA.     

A general purpose method for extracting RNA from Dictyostelium cells

Here we present a protocol for the extraction of RNA from Dictyostelium discoideum. Dictyostelium is a social amoeba that undergoes a basic developmental program, and therefore analysis of RNA levels over a time course is a commonly used technique. This procedure is similar to other guanidine thiocyanate-based methods; however, it has been adjusted because of the large quantities of carbohydrate and nucleases found in Dictyostelium cells. After cell lysis and phenol:chloroform extraction, the resulting high-quality RNA isolated with the described protocol allows the molecular genetic analysis of wild-type and genetically modified cells. The purified RNA can be used for analyses such as northern blotting, RT-PCR and microarrays. This procedure requires approximately 2 h to complete.     

A simple procedure for large-scale preparation of pure plasmid DNA free from chromosomal DNA from bacteria

A very simple, inexpensive procedure for preparing pure plasmid DNA from bacteria is described. In this method, lysozyme-induced spheroplasts are made in presence of 833 micrograms/ml of ethidium bromide which are then lysed by a mixture of Brij 58 and sodium deoxycholate, and the lysate is centrifuged at 48,000 g for 25 min whereby about 99.9% of total chromosomal DNA is pelleted. From the supernatant containing plasmid DNA, the proteins are removed by phenol extraction and the major part of RNA by CaCl2 precipitation, and finally the small amount of residual RNA is removed by RNase treatment. The average yield of pBR322 DNA from 1 liter of amplified culture by this procedure is 2 to 2.5 mg and the preparation is highly pure, containing only about 0.005% of total yield as chromosomal DNA contaminant. Moreover, the substrate activity and the transforming ability of the plasmid DNA prepared by this method remain unaffected.     

A method for the preparation of high molecular weight yeast DNA

A method is described for the isolation of high molecular weight DNA in solution using the principles that have allowed electrophoresis of chromosome-sized DNA in pulse field gradient electrophoresis. Stationary phase yeast cells are converted to spheroplasts by the action of zymolyase in 1 M sorbitol. In the presence of EDTA and sodium lauroyl sarcosinate, proteins are digested with proteinase K. DNA is extracted with phenol and chloroform, and high molecular weight DNA is collected by ethanol precipitation. RNA is removed by RNase digestion of the redissolved pellet, and RNase is removed by chloroform extraction followed by a second ethanol precipitation. The method is rapid and gives a high yield of DNA that is readily digestible by restriction endonucleases.     

Purification of human genomic DNA from whole blood using sodium perchlorate in place of phenol

We have developed a new, rapid method for the extraction of human genomic DNA from whole blood samples. Traditionally, genomic DNA has been extracted from blood by overnight proteinase K digestion of lysed peripheral lymphocytes followed by phenol/chloroform extraction. In addition to being time consuming, the use of phenol involves inherent risks due to the toxic nature of the reagent. Our method for the extraction of DNA from whole blood uses sodium perchlorate and chloroform instead of phenol with a significant time savings realized as well as fewer hazards to the technician. Furthermore, DNA prepared by this new method is an excellent substrate for restriction endonuclease digestion and Southern hybridization analysis.     

一种高效经济的高质量植物RNA提取方法

建立了一种高效经济的植物RNA提取方法.在提取缓冲液中加入蔗糖、氯化钾和镁离子以提供对RNA分子的保护.破碎后的细胞于提取缓冲液中裂解后,用酚/氯仿变性并去除内源RNA酶和其他蛋白质,而后用pH 5.6 的NaAc沉淀RNA.用该方法提取RNA的得率较高,经电泳检测,RNA的完整性很好.RNA印迹分析和RT-PCR也都得到很好的结果.该方法还使实验成本大大降低.     

Isolation of polysaccharide-free DNA from plants

A quick procedure for the isolation of polysaccharide-free DNA from different plant species and cell suspension or callus cultures is described. The originality of the method lies in the use of a mixture of glycoside hydrolases that leads, after phenol and chloroform extraction, to the isolation of pure DNA without any polysaccharide contamination. The highly purified DNA can be used for nucleotide analysis by HPLC, RFLP analysis and PCR amplification.