DNA isolation protocol for seaweeds

We report a DNA isolation protocol for red seaweeds. Recovering DNA of high quality and quantity is a prerequisite for ensuring suitable applications, such as polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis, and sequencing. Isolation of DNA from seaweeds has proven difficult because of coprecipitation of polysaccharides. Our process minimizes this contamination, which is mostly due to the highly hydrocolloidal content of algal cell walls. This protocol, using 2 steps, is based on a preliminary enzymatic digestion of cell wall with specific enzymes (Novozymes) followed by centrifugation, allowing isolation of DNA on the pellet. This provides a higher yield of DNA, in the range of 40 μg (Palmaria palmata) and 18 μg (Gracilaria verrucosa) from 50 mg of fresh frozen pellet.     

Design of a microaerobically inducible replicon for high-yield plasmid DNA production

A pUC-derived replicon inducible by oxygen limitation was designed and tested in fed-batch cultures of Escherichia coli. It included the addition of a second inducible copy of rnaII, the positive replication control element. The rnaII gene was expressed from P_trc and cloned into pUC18 to test the hypothesis that the ratio of the positive control molecule RNAII to the negative control element, RNAI, was the determinant of plasmid copy number per chromosome (PCN). The construct was evaluated in several E. coli strains. Evaluations of the RNAII/RNAI ratio, PCN and plasmid yield normalized to biomass (Y_pDNA/X) were performed and the initial hypothesis was probed. Furthermore, in high cell-density cultures in shake flasks, an outstanding amount of 126 mg/L of plasmid was produced. The microaerobically inducible plasmid was obtained by cloning the rnaII gene under the control of the oxygen-responsive Vitreoscilla stercoraria hemoglobin promoter. For this plasmid, but not for pUC18, the RNAII/RNAI ratio, PCN and Y_pDNA/X efficiently increased after the shift to the microaerobic regime in fed-batch cultures in a 1 L bioreactor. The Y_pDNA/X of the inducible plasmid reached 12 mg/g at the end of the fed-batch but the original pUC18 only reached ca. 6 mg/g. The proposed plasmid is a valuable alternative for the operation and scale-up of plasmid DNA production processes in which mass transfer limitations will not represent an issue.     

A simple protocol for isolation of fungal DNA

Genomic DNA was isolated from as little as 2 mg dry biomass of Magnaporthe grisea by microwave treatment within 30 s. The quantity of DNA was good enough for PCR analysis and Dot blot hybridization. This technique can be used for various studies, such as DNA fingerprinting to study the population structure of the phytopathogen in different regions, and for a quick screening of M. grisea transformants.     

A novel method for rapid isolation of plasmid DNA

A new disposable chromatographic column, pZ523, has been developed for separating plasmid DNA from bacterial chromosomal DNA. Use of pZ523 spun columns eliminates the need for ethidium bromide-cesium chloride density gradients which require long centrifugation times. pZ523 purified plasmids have been shown to be of purity suitable for restriction analysis, ligation, transfection of mammalian cells and transformation of bacteria. Unlike the traditional ultracentrifugation method, pZ523 offers an extremely rapid alternative method for purifying large amounts of plasmid DNA (2.5 mg to 4.5 mg) from cleared bacterial lysates in only 25 minutes.     

DNA isolation protocol for red seaweed (rhodophyta)

We report a DNA isolation protocol for red seaweed. The method is a modification of the Dellaporta et al. (1983) protocol for land plants. Our simplified version can be used to process large sample numbers and to minimise polysaccharide co-isolation. The protocol was applied to 12 red seaweed species as well as one green alga and one land plant. The protocol yields about 5 μg of high molecular weight DNA from 10 mg of dried material, with no RNA. No sign of degradation was observed after agarose gel electrophoresis for both freshly extracted DNA and DNA stored for 18 months at 4°C. DNA isolated by our protocol was suitable for genomic library construction (tested for one species), endonuclease restriction, and PCR amplification for all species.     

碱裂解提取质粒DNA的改进

碱裂解提取质粒DNA是分子生物学实验中常用的方法,但通常方法所提取的质粒往往含有大量的RNA和其他杂质.本文适时加入较高浓度的RNA酶和适当延长冰浴时间,结果得到了几乎没有RNA和其他杂质的高纯质粒DNA,不仅达到了分子生物学实验要求,而且可用作抗原检测抗dsDNA抗体.该法操作简单、经济、实用.     

An efficient,simple and high throughput protocol for cotton genomic DNA isolation

Cotton is a stubborn plant for genomic DNA isolation due to its high-level of polyphenolics, polysaccharides and secondary metabolites. Genomics and molecular biology studies require high quality and large quantity of DNA. We have standardized an efficient miniprep protocol for cotton genomic DNA isolation, which not only provides higher yield from 800 to 1400 µg of DNA from 200 to 300 mg of fresh leaf tissue but also provide excellent purity. The DNA is amenable to all elementary enzymatic preparations, PCR techniques, Southern blotting and to high end genomic studies. The technique does not require liquid nitrogen, needs small amount of sample, less time, fewer chemicals and one can process up to 100 samples per day. The genomic DNA extracted was good for transgenic event characterization and marker assisted selection.     

Large-scale isolation of plasmid DNA using cetyltrimethylammonium bromide

A rapid procedure for the large-scale isolation of plasmid DNA is described. The method utilizes cetyltrimethylammonium bromide to precipitate the plasmid following extraction of DNA by lysozyme digestion and boiling. The plasmid is then purified by passing through the spin column pZ523. The purity and yield of the plasmid obtained with this method is similar to that isolated by cesium chloride-ethidium bromide gradient centrifugation. The method does not involve any phenol-chloroform extractions and takes five to six hours for completion after growth of the bacterial cells. The plasmid obtained is amenable to digestion with various restriction endonucleases, can be used for cloning with high efficiency and is also suitable as template for dideoxy sequencing.     

A cost-effective plasmid purification protocol suitable for fluorescent automated DNA sequencing

A cost-effective, reliable, and reproducible method has been developed to produce good-quality, double-stranded plasmid DNA for automated sequence analysis. The method incorporates modifications to a previously described plasmid-purification protocol used in manual sequencing. The quality of the DNA produced from the present protocol is suitable for automated fluorescent sequencing. Using a dye-terminator sequencing protocol, most runs using plasmid DNA prepared using this protocol produced over 700 bases with greater than 99% base-calling accuracy.     

An efficient method for isolation of plasmid DNA from methylotrophic bacteria

A method, suitable for the isolation of closed circular plasmid DNA from methylotrophic bacteria is described. Improvement of cell lysis was achieved by butanol extraction of cells before application of the lytic agent. Using this method, cryptic plasmids of 7.8, 14, 36 and 200 kb were purified from soil-isolated methylotrophs.     

A rapid method for the isolation of plasmid DNA from bacteria

Summary A rapid method is described for the isolation of plasmid DNA from Escherichia coli and Pseudomonas putida. The effect of heating the cell preparation during plasmid extraction is discussed in relationship to the final plasmid yield.     

Rapid isolation of high quality, multimeric plasmid DNA using zwitterionic detergent

Purification of plasmid DNA from bacteria is an essential tool in recombinant DNA technology and has become an essential task in laboratories to industries. Moreover, the recent progress of "Gene therapy" and "Genetic vaccination" also demands production of pharmaceutical grade plasmid DNA in 'kilogram' level. Despite existence of a number of purification protocols, all most all have been originated from a pioneering work [Birnboim, H.C., Doly, J., 1979. A rapid extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523] and so suffer from one or more drawbacks, such as purification time, purity or quantity of isolated plasmid DNA. Here, we have reported an innovative approach for isolation of highly pure and functional plasmid DNA in significant amount, based on generation of "soft protein aggregate" with the help of zwitterionic detergents and alkali. Solibilized proteins and RNA could be removed by a simple and mild washing with Tris buffer of low ionic strength and multimeric plasmid DNA could be eluted in a single step from the protein aggregate. Additionally, isolated plasmid DNA could easily be digested by restriction enzymes and had high functionality in protein expression. Thus, considering both its remarkable simplicity and efficiency in producing sufficiently pure plasmid DNA, the new strategy would emerge a useful tool in modern recombinant technology and therapeutic applications.