scholarly journals Filter paper-based spin column for low throughput nucleic acid purification

2018 ◽  
Author(s):  
Rui Shi ◽  
Ramsey S. Lewis ◽  
Dilip R. Panthee
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Filter Paper ◽  
Genomic Dna ◽  
Total Rna ◽  
Binding Material ◽  
Pcr Product ◽  
Spin Column ◽  
Nucleic Acid Purification ◽  
Commercial Kits

AbstractWe describe a method of recharging used spin column or assembling homemade spin column using filter paper as binding material for low throughput nucleic acid purification. We evaluated the efficiency of filter paper based spin columns in the purification of different type of nucleic acids. For instance, by following protocols of respective commercial kits, we found that filter paper to be a useful binding material for purification of many types of nucleic acids, including plant genomic DNA, plant total RNA, PCR product, and DNA from agarose gels. We also found that filter paper has a weak binding affinity to plasmid DNA in tested miniprep protocols. Also, we present the protocols of using filter paper recharged spin column or homemade spin column for low throughput purification of plant genomic DNA and plant total RNA with commercial kit buffer leftover and less expensive homemade buffer.

Research note: Rapid isolation of total RNA and genomic DNA from Hakea actities

2002 ◽  
Vol 29 (8) ◽  
pp. 1015 ◽  
Author(s):  
Michael G. Mason ◽  
Susanne Schmidt
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Genomic Dna ◽  
Rna Extraction ◽  
Nutrient Concentrations ◽  
Cluster Roots ◽  
Acid Extraction ◽  
Native Plant ◽  
Nucleic Acid Extraction ◽  
Total Rna

Tissues of the Australian native plant species Hakea actities (Proteaceae) contain numerous metabolites and structural compounds that hinder the isolation of nucleic acids. Separate RNA and genomic DNA extraction procedures were developed to isolate high quality nucleic acids from H. actities. Total RNA was extracted from leaves, roots and cluster roots of H. actities grown in low nutrient levels. Cluster root formation in H. actities only occurs when the plants are grown in low nutrient concentrations. However, under these conditions, nucleic acid extraction becomes increasingly difficult. The new procedures are faster than many of the published nucleic acid extraction protocols, and avoid the use of hazardous chemicals. The RNA extraction method was used successfully on another Australian species and a crop species, suggesting that the procedure is useful for molecular studies of a broad range of plants.

scholarly journals Validating DNA Extraction Protocols for Bentonite Clay

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Katja Engel ◽  
Sara Coyotzi ◽  
Melody A. Vachon ◽  
Jennifer R. McKelvie ◽  
Josh D. Neufeld
Keyword(s):  
Microbial Community ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Bentonite Clay ◽  
Dna Recovery ◽  
Blocking Agents ◽  
Clay Matrix ◽  
Microbial Dna

ABSTRACT Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material “spiked” with Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination. IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.

scholarly journals Deteksi Staphylococcus aureus dan Staphylococcus sp. Secara Langsung Dari Susu Segar Kambing Peranakan Etawa dengan Teknik PCR

2020 ◽  
Vol 38 (2) ◽  
pp. 168
Author(s):  
Fatkhanuddin - Aziz ◽  
Fajar Budi Lestari ◽  
Sarah Nuraida S. ◽  
Endah Purwati ◽  
Siti Isrina Oktavia Salasia
Keyword(s):  
Staphylococcus Aureus ◽  
Nucleic Acid ◽  
23S Rrna ◽  
Food Borne ◽  
Spin Column ◽  
Nucleic Acid Purification

Genus Staphylococcus merupakan salah satu patogen bakteri penyebab mastitis yang menyebabkan kerugian ekonomi pada kambing Peranakan Etawa. Diantara Staphylococcus sp. yang dapat tumbuh dengan baik dalam susu segar, diketahui Staphylococcus aureus dapat membahayakan kesehatan manusia yang mengkonsumsi (food borne disease) karena kemampuannya dalam memproduksi enterotoksin yang tahan terhadap enzim pencernaan maupun pemanasan. Tujuan penelitian ini adalah mendeteksi Staphylococcus sp. dan S. aureus secara langsung dari susu kambing peranakan etawa dengan teknik PCR.Metode yang dilakukan adalah mengekstraksi DNA dari 60 sample susu segar dengan prinsip spin column-based nucleic acid purification dan kemudian dilakukan amplifikasi gen spesifik 23S rRNA Staphylococcus sp. dan S. aureus. Hasil PCR diketahui 37 (61%) sampel susu positif mengandung Staphylococcus sp. dan hanya 1 (1,6%) sampel mengandung S. aureus. Metode deteksi dengan PCR dapat digunakan untuk mendeteksi kontaminan Staphylococcus sp. dan S. aureus dengan waktu yang singkat

scholarly journals Simple, inexpensive and RNase-free purification of plasmid DNA by fractional precipitation with isopropanol

BioTechniques ◽  
2021 ◽  
Author(s):  
Viiu Paalme ◽  
Mart Speek
Keyword(s):  
Quality Control ◽  
Column Chromatography ◽  
Plasmid Dna ◽  
Fractional Precipitation ◽  
Alkaline Lysis ◽  
Bacterial Extract ◽  
Total Rna ◽  
Spin Column ◽  
Commercial Kits

We present a modified alkaline lysis method for purification of plasmid DNA (pDNA) from bacterial extract using fractional precipitation with isopropanol (FPI). This method includes two successive precipitations with 0.33 and 0.36 volumes of isopropanol and separates pDNA from total RNA and most of the lipopolysaccharides. Using different quality control tests, we demonstrate that plasmids purified with FPI show superior quality compared to plasmids prepared with commercial kits based on spin-column chromatography.

scholarly journals Total RNA Extraction from the Aromatic Phalaenopsis bellina, Endemic Orchid in Sabah, Borneo

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Ahmad Asnawi MUS ◽  
Jualang Azlan GANSAU ◽  
Nor Azizun RUSDI
Keyword(s):  
Genomic Dna ◽  
Molecular Level ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Structural Instability ◽  
Total Rna ◽  
Isolation Methods ◽  
Commercial Kits ◽  
Yield And Purity ◽  
Total Rna Extraction

Phalaenopsis bellina is an attractive orchid due to its unique appearance and distinctive floral fragrance. Many past studies on this plant focused on the plant at the molecular level; however, this requires sufficient quantities of high-quality P. bellina RNA. RNA is more delicate to manipulate than DNA due to its structural instability and its vulnerability to various secondary metabolites, such as polyphenols and polysaccharides. Therefore, in this study, 4 RNA isolation methods, a modified phenol-chloroform method and 3 commercial kits (Vivantis, Novogene, and Analytik Jena) were used on the leaves and flowers of P. bellina for comparison. The yield and purity of the total RNA were determined using spectrophotometry. The results showed that the total RNA isolated using the modified phenol-chloroform method had the highest yield (1223.75±68.51 ng/µL) and purity compared to the 3 commercial kits, with an OD260/280 value of 2.07 and an OD260/230 value of 2.26, respectively. In particular, the isolated RNA did not show any detectable genomic DNA contamination or other impurities. The RNA isolated using the phenol-chloroform method was also evaluated by electrophoresis, reverse transcription, and PCR. The results indicated that the phenol-chloroform method appears to be superior for total RNA extraction. Thus, this developed method is proven to be suitable for the RNA extraction of plants rich in polysaccharides and polyphenols and is amenable for future molecular studies on P. bellina.

Picloram Sensitivity and Nucleic Acids in Plants

Weed Science ◽  
1970 ◽  
Vol 18 (1) ◽  
pp. 1-4 ◽  
Author(s):  
S. S. Malhotra ◽  
J. B. Hanson
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Herbicide Resistance ◽  
Dna Content ◽  
Acid Metabolism ◽  
Nucleic Acid Metabolism ◽  
Sensitive Species ◽  
Total Rna ◽  
Resistant Species ◽  
Rna And Dna

The changes in the nucleic acid metabolism were studied in plants susceptible and resistant to 4-amino-3,5,6-trichloropicolinic acid (picloram). The total RNA and DNA content of the tissue correlated inversely with the herbicide resistance; the resistant plants were low in nucleic acids, whereas sensitive plants were high. The increase in the nucleic acids of the sensitive species 24 hr after picloram treatment appeared to be associated with lower levels of ribonuclease and deoxyribonuclease. The inability of the resistant species to make more RNA may be associated with high levels of nucleases in the tissue.

scholarly journals Evaluating the efficiency of ethanol precipitation method in purification of gDNA and PCR product

2019 ◽  
Vol 32 ◽  
pp. 276-281
Author(s):  
Labeed A. Al-Saad ◽  
Ayoob R. Al-Zaalan
Keyword(s):  
Nucleic Acid ◽  
Standard Method ◽  
Genomic Dna ◽  
Sodium Acetate ◽  
Product Yield ◽  
Precipitation Method ◽  
Pcr Product ◽  
Dna Purity ◽  
Yield And Purity ◽  
Specific Amount

A numerous clean-up methods of nucleic acid were developed to achieve the requirements of downstream reactions like PCR and sequencing. The methods were varied in their mechanism, efficiency of purification and final product yield. The present study evaluated the efficiency of ethanol-sodium acetate (EOH-NaOAc3) precipitation method in purification of nucleic acid to satisfy downstream reactions requirements. The yield and purity of nucleic acid were considered as the main standard parameters to estimate the efficiency of method. Geneaid gel extraction kit DF100 was considered as a standard method for comparison. The results of methods comparison revealed that EOH-NaOAc3 method was significantly (P=0.000) surpassed the kit method in the yield of purified PCR product (93.24 and 18.37 ng/µl) with no significant differences (P=0.239) in quality (Absorbance (A260/280+) = 1.816 and 1.843) respectively. To determine the productivity of EOH-NaOAc3 method, a specific amount of genomic DNA (G-DNA) (187.93 ng/ µl) was processed and the results showed that EOH-NaOAc3 method was efficiently conserved 89.6% of total processed G-DNA (168.51 ng/µl) accompanied by significant (P=0.03) elevation of DNA purity (A260/280, 3.07 – 2.53).

scholarly journals Evaluation of commercial methods to separate nucleic acids from intestinal tissues of pigs for diagnosis of porcine epidemic diarrhea

2019 ◽  
Vol 10 (4) ◽  
pp. 477-483
Author(s):  
D. М. Masiuk ◽  
V. S. Nedzvetsky ◽  
A. V. Kokariev ◽  
O. V. Danchuk ◽  
T. O. Vasilenko ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Solid Phase ◽  
Rna Extraction ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Viral Dna ◽  
Tissue Mass ◽  
Porcine Epidemic Diarrhea ◽  
Commercial Kits

The article presents the results of evaluating commercial methods for extracting nucleic acids from pig intestinal tissues for the diagnosis of PED. The study was based on samples of small intestine tissues and faeces from 3–5 day old pigs which died from PED. Nucleic acid extraction was performed using commercial kits with different nucleic acid separation strategies based on: silicon-sorbent; silicate membrane fixed in a microcentrifuge column and magnetic balls. The studies were conducted in two stages. The first was a comparison of the results of the amplification of the obtained nucleic acid extracts from the homogenate of the intestines of piglets by using the above-mentioned commercial kits for the extraction of nucleic acids. For this purpose, samples of homogenate were used which in weight corresponded to the guideline for the application of the test kits. The second step was directed to determining the efficiency of extraction of DNA and RNA from homogenate samples with a weight of 10, 50, 100 and 200 mg. Determination of the optimal methodological strategy of nucleic acid extraction for the diagnosis of porcine epidemic diarrhea by PCR has been investigated. The results of the PCR studies of RNA of the PED virus and a unique pig DNA fragment indicate that the extraction of nucleic acids by commercial kits has different levels of efficiency and depends on different factors. According to the research, it was found that the most important of them are the adsorption capacity of the solid-phase sorbent, its configuration and nature, which binds RNA and DNA molecules, the type of sample from which extraction takes place, its volume, or the tissue mass used for extraction. Based on the obtained results, it has been found that the most effective PED virus RNA extraction is by “ArtBioTech”, “Bio Extract Column”, and “Viral DNA/RNA Extraction Kit”, and pig genomic DNA extraction by the “ArtBioTech” and “Viral DNA / RNA extraction Kit”.

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