Although we found that filter paper-based spin columns do accommodate an in-column DNA digestion protocol as suggested in the Qiagen kit manual, we recommend treating RNA elution using a DNA-free kit of Invitrogen or similar kits to eliminate remaining DNA. Nucleic acid purification using filter paper is mainly associated with cellulose, which appears to share similar features as silica-based materials in the binding of nucleic acids in chaotropic condition.
However, only secondary fibril-associated cellulose within filter paper was found to isolate nucleic acids [ 5 ]. We found filter paper to be more efficient in the purification of high molecular weight plant genomic DNA, which is consistent with the report from Promega's Paramagnetic cellulose, which improves DNA yield for plant species [ 6 ]. Also, we found that filter paper is less efficient for plasmid DNA purification following a miniprep protocol. Reasons for this observation are unclear, however.
These results suggest that cellulose-based material may bear some differences in mechanisms as compared silica-based material in binding and elution of nucleic acids. Unlike the silica-based nucleic acids purification process that has been extensively studied [ 26 ], there are few reports investigating the interaction between cellulose and nucleic acids [ 5 , 13 ]. Therefore, more studies are needed to facilitate application of filter paper-based nucleic acid purification.
We found filter paper to be an appropriate substitute for silica-based materials for purification of nucleic acids from various sources. Filter paper can be easily adopted to recharge used spin columns or to prepare homemade spin columns for low throughput applications using commercial kit buffers or homemade buffers to reduce laboratory costs.
Filter paper can therefore be an important component for nucleic acid purification in molecular biology laboratories. D Prepare homemade filter paper-based spin column prepared using 0. The right lane is bp DNA marker two strong bands are 1kb and bp in size. We are thankful to Ann Piotrowski for her help in planting tomato plants in the greenhouse and field, Prof. Wei Shi for access to the NanoDrop instrument, and Prof.
Ronald Sederoff for help on revising the manuscript. The work was partially supported by North Carolina State University in the form of enhancing funding to Prof. Ramsey S. Dilip R.
Panthee, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Center for Biotechnology Information , U.
PLoS One. Published online Dec 7. Maxim Antopolsky, Editor. Author information Article notes Copyright and License information Disclaimer. Competing Interests: The authors have declared that no competing interests exist. Received Aug 9; Accepted Nov Copyright notice. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
The work is made available under the Creative Commons CC0 public domain dedication. This article has been cited by other articles in PMC. Associated Data Supplementary Materials S1 Fig: Illustration of assembling different formats of filter paper-based spin column.
Abstract We describe herein a method of recharging used commercial spin columns or assembling homemade spin columns using filter paper as binding material for cost-effective, low throughput nucleic acid purification. Introduction Nucleic acid purification is the starting point for many applications in molecular biology [ 1 , 2 ]. Open in a separate window. Fig 1. Recharging commercial spin columns and homemade spin columns using filter paper.
Purification of plant genomic DNA Plant genomic DNAs were purified using filter paper-based spin columns following the modified protocol of the Qiagen DNeasy Plant mini kit Qiagen DNeasy plant handbook, March version or an in-house protocol using homemade buffers described by Lemke et al [ 9 ]. Evaluation of purified nucleic acids DNA yield and quality were evaluated using 1. Results Recharged spin columns and homemade spin columns using filter paper Our efforts to prepare a filter paper-based spin column were initiated by recharging used commercial spin columns with filter paper disc s.
Evaluation of filter paper in the purification of nucleic acids from various sources Our initial experiments suggested that filter paper may function in commercial kits for nucleic acid purification.
Fig 2. The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. Purification of plant DNA or RNA using filter paper-based spin columns with commercial kit buffer or homemade buffer We expected that filter paper-based spin columns could be used as a substitute for commercial spin columns using the remnant buffer of commercial kit to save financial resources. Fig 3. Evaluation of purification of tobacco genomic DNA and total RNA using filter paper-based spin columns with respective Qiagen kit buffers and homemade buffers.
Discussion Development of filter paper-based spin columns is useful for nucleic acid purification because spin columns are widely used and associated methodology aligns with conventional desktop centrifuge equipment for low throughput bench-scale experiments involving nucleic acids. Conclusions We found filter paper to be an appropriate substitute for silica-based materials for purification of nucleic acids from various sources.
Supporting information S1 Fig Illustration of assembling different formats of filter paper-based spin column. TIF Click here for additional data file. Acknowledgments We are thankful to Ann Piotrowski for her help in planting tomato plants in the greenhouse and field, Prof.
Data Availability All relevant data are within the manuscript and its Supporting Information files. References 1. J Biomed Biotechnol. Thatcher SA. Clinical chemistry. McCormick RM. A solid-phase extraction procedure for DNA purification. Anal Biochem. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. Su X, Comeau AM. Cellulose as a matrix for nucleic acid purification. Applications in Plant Sciences.
Complete decontamination and regeneration of DNA purification silica columns. Anal biochem. A simple, inexpensive and environmentally friendly method for high throughput DNA extraction from grapevine Vitis spp.
Nucleic acid-free matrix: regeneration of DNA binding columns. An advantage of silica membrane spin columns relative to glass beads Vogelstein and Gillespie, is that they help decrease shearing of DNA fragments that are larger than 3 to 10 kb. Binding Mechanism The principle of silica matrices purification is based on high affinity of negatively charged DNA backbone towards the positively charged silica particles Esser et al.
DNA binds to silica through hydrogen-binding interacation with an underivatised hydrophilic matrix provided by silica, under concentrated chaotrophic salt conditions usually sodium iodide, sodium perchlorate, guanidinium thiocyanate Berensmeier, DNA is tightly bound, and extensive washing removes all contaminants.
The adsorption of plasmid and chromosomal DNA on microcrystalline silica surface and the effect of ionic strength, temperature, pH, DNA size and conformation on the adsorption phenomenon were reported by Melzak et al. It was inferred from the isotherms that i shielded intermolecular electrostatic forces, ii dehydration of the DNA and silica surface and iii intermolecular hydrogen bond formation in the DNA silica contact layer are the major contributing driving force for adsorption.
Under high salt concentrations, nucleic acids selectively bind to silica membranes while other contaminants, mainly proteins, pass through the membranes.
By and large, guanidinium thiocyanate and guanidinium hydrochloride are used for binding nucleic acids to silica membranes. Guanidinium thiocyanate at a concentration of 4M ot 6M works best, while guanidinium hydrochloride is used at higher concentrations in excess of 6M. In order to control pH of the binding reagent, sodium acetate and Tris-HCl buffers, ranging from pH 6.
The binding efficiency is significantly improved in the presence of ethanol. Sodium iodide and sodium perchlorate are also used to a lesser extent for nucleic acid binding. Guanidinium thiocyanate is an excellent protein denaturant and hence very effective in inactivating nucleases. However, detergents such as sodium dodecyl sulfate is not compatible with guandinium thiocynate, but works well with guanidinium hydrochloride.
Spin column-based nucleic acid purification is defined as a solid-phase extraction procedure to purify nucleic acids quickly. This process depends on the fact that nucleic acids tend to bind to the solid phase of silica under specific conditions. The process basically involves 4 steps:. Spin Column tubes are specially produced to be used in purification, isolation, and separation of nucleic acids and have a diverse range of applications starting from DNA to RNA, plasmid to viral and genomic DNA, regular PCR purification to the next-generation sequencing sample preparation.
Your email address will not be published. Skip to content Monday, October Lab techniques Biomall August 28, 0. What are spin columns? Different types of spin columns 1. Anion exchange spin column: These spin columns help in the transfection grade plasmid DNA purification. Some features of anion exchange spin columns are: Fast and simple: Membrane-based spin design removes column packing Convenient and flexible: Centrifugal format empowers advantageous handling of different samples in parallel Robust: These do not crack or run dry Low bed volume: working with low buffer volume is possible due to small membrane adsorber bed volumes that results in concentrated elution portions 3.
Filter paper based spin column: Filter paper made from cellulose fiber is a potential substitution for silica material in a spin column for high throughput purification. Polyethylene filter spin column: These types of spin columns are appropriate for coarse filtration along with particle removal.
0コメント