Extracting DNA from submerged pine wood

Genome ◽  
2004 ◽  
Vol 47 (5) ◽  
pp. 994-997 ◽  
Author(s):  
M Megan Reynolds ◽  
Claire G Williams
Keyword(s):  
Dna Extraction ◽  
Dna Analysis ◽  
Pcr Amplification ◽  
Pinus Palustris ◽  
Proteinase K ◽  
High Molecular Weight Dna ◽  
Bisphosphate Carboxylase ◽  
Extraction Protocol ◽  
High Sequence Homology ◽  
Reaction Sequencing

A DNA extraction protocol for submerged pine logs was developed with the following properties: (i) high molecular weight DNA, (ii) PCR amplification of chloroplast and nuclear sequences, and (iii) high sequence homology to voucher pine specimens. The DNA extraction protocol was modified from a cetyltrimehtylammonium bromide (CTAB) protocol by adding stringent electrophoretic purification, proteinase K, RNAse, polyvinyl pyrrolidone (PVP), and Gene Releaser®. Chloroplast rbcL (ribulose-1,5-bisphosphate carboxylase) could be amplified. Nuclear ribosomal sequences had >95% homology to Pinus taeda and Pinus palustris. Microsatellite polymorphism for PtTX2082 matched 2 of 14 known P. taeda alleles. Our results show DNA analysis for submerged conifer wood is feasible.Key words: conifers, wood, polymerase chain reaction, sequencing.

scholarly journals Quality and Quantity of Extracted Deoxyribonucleic Acid (DNA) from Preserved Soft Tissues of Putrefied Unidentifiable Human Corpse

2014 ◽  
Vol 6 (01) ◽  
pp. 031-035 ◽  
Author(s):  
Shashank Pooniya ◽  
Sanjeev Lalwani ◽  
Anupuma Raina ◽  
Tabin Millo ◽  
Tirath Das Dogra
Keyword(s):  
Dna Extraction ◽  
Dna Analysis ◽  
Soft Tissues ◽  
Pcr Amplification ◽  
Climatic Conditions ◽  
High Molecular Weight Dna ◽  
Human Corpse ◽  
Multiple Loci ◽  
Polymerase Chain ◽  
The Brain

ABSTRACT Context: The appropriate collection and preservation of soft tissues from putrefied unidentifiable human corpse for the purpose of identification using DNA profiling technique is critically important especially in developing countries like India having different levels of health-care set ups with largely varying facilities and varying climatic conditions. Aims: The present study was carried out, mainly focusing on quality and quantity of extracted DNA from the soft tissues of putrefied unidentifiable human corpse stored upto 4 weeks at 4°C and at −80°C for DNA analysis. Materials and Methods: The present study was conducted on 16 different putrefied unidentifiable human corpses after getting approval from institutional ethical committee. Around 2 g of four different tissues (brain, kidney, heart and muscle) were collected and preserved for one month followed by DNA extraction using the organic method, the quality and quantity of high molecular weight-DNA was estimated using the spectrophotometer and gel electrophoresis. Further, the amplification polymerase chain reaction (PCR) was also performed (AmpFLSTR® Indentifiler™ PCR Amplification kit for multiple loci, of Applied Biosystems, Lab India) and was checked using continuous PAGE. Results: The yield of DNA was significantly higher at −80°C for all the four tissues collected and was best for brain followed by heart, kidney and worst for muscles in all cases. Conclusions: It is suggested that the brain tissue preserved at −80°C is the best among soft issues for DNA extraction. Refrigeration or deep freezing facility should be available at all the centers.

scholarly journals Optimization of genomic DNA extraction protocol for black wattle

Agrarian ◽  
2020 ◽  
Vol 13 (49) ◽  
pp. 323-329
Author(s):  
Yasmin Imparato Maximo ◽  
Angela Cristina Ikeda ◽  
Paulo César Flôres Júnior ◽  
Giovana Bomfim De Alcantara ◽  
Antonio Higa
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Proteinase K ◽  
Genomic Dna Extraction ◽  
Extraction Protocol ◽  
Black Wattle

Considerando-se que a atual tendência do melhoramento florestal é a integração das técnicas clássicas com as de análise genética molecular, faz-se necessária a obtenção de protocolos de extração de DNA genômico ajustados a cada espécie estudada. O objetivo do trabalho foi determinar o efeito de diferentes adaptações no protocolo de extração de DNA genômico CTAB para acácia-negra. Foram testados diferentes componentes na fase de extração orgânica: clorofórmio, fenol e proteinase K, além da aplicação de RNase após a fase de precipitação e limpeza do DNA. Também, foi investigada a eficiência destes tratamentos em amostras de folíolos frescas ou armazenadas em baixa temperatura durante sete dias. Foi verificada a presença de DNA de todas as amostras submetidas à extração pelo protocolo de CTAB com os diferentes tratamentos. O tempo de armazenamento das amostras não influenciou na integridade do DNA, entretanto, foi possível observar que a adição de RNase melhorou a qualidade do DNA extraído. Deste modo, sugere-se a utilização do protocolo CTAB com uso de clorofórmio e RNase, com amostras frescas ou armazenadas em baixas temperaturas.

Sample collection and eDNA extraction from Sterivex filter units v1

2021 ◽  
Author(s):  
Oscar E Chiang ◽  
Pedro Inostroza
Keyword(s):  
Dna Extraction ◽  
Environmental Samples ◽  
Dna Analysis ◽  
Water Systems ◽  
Sample Collection ◽  
Extraction Protocol ◽  
Water Collection ◽  
Sterile Filter

The following workflow covers several steps in the DNA analysis of environmental samples, from the water collection to the analysis back in the lab. The samples can be taken from several water systems (i.e. sea, lakes, rivers, streams) and collected in triplicate (1 L) in Sterivex sterile filter units (Merck, cat. no. SVGP01050). The DNA extraction protocol modifies the Dneasy PowerWater Sterivex kit (Qiagen, cat. no. 14600-50-nf).

scholarly journals EQUAL-qual: A European Program for External Quality Assessment of Genomic DNA Extraction and PCR Amplification

2007 ◽  
Vol 53 (7) ◽  
pp. 1349-1357 ◽  
Author(s):  
Claudio Orlando ◽  
Paolo Verderio ◽  
Ronald Maatman ◽  
Jan Danneberg ◽  
Simon Ramsden ◽  
...  
Keyword(s):  
Quality Assessment ◽  
Dna Extraction ◽  
Dna Analysis ◽  
External Quality Assessment ◽  
Clinical Chemistry ◽  
Pcr Amplification ◽  
Molecular Techniques ◽  
The European Union ◽  
External Quality ◽  
Pcr Efficiency

Abstract Background: Despite the rapid transition into routine clinical practice of molecular techniques based on PCR, external quality assessment (EQA) is still not widely available. The European Union and European Communities Confederation of Clinical Chemistry have supported the EQUAL project as a series of 3 different EQA programs for the assessment of molecular methods independently from analytes. We present the results from the EQUAL-qual program designed to evaluate the analytical aspects of DNA analysis by means of a conventional qualitative PCR experiment. Methods: The EQUAL-qual program provided DNA, blood samples, and primer sets to participant laboratories to assess DNA extraction and PCR amplification. We have developed statistical procedures to identify laboratories performing poorly in DNA extraction (quality and quantity), PCR efficiency, and data interpretation after electrophoresis. Results: An application to participate was obtained from 213 laboratories (from 25 countries), and 175 (82%) of laboratories submitted results for assessment. Questionable results in terms of quality and/or quantity of DNA derived from blood extractions were returned by 27% of laboratories (46 of 166). PCR efficiency showed high variability, with 3% of laboratories (5 of 163) showing a consistently low rate of amplification and 10% (18 of 175) not reporting the expected number of bands of the amplified targets. Conclusions: The results showed considerable variability in all phases of the experiment. The approach confirms the validity of EQA as a method for evaluating analytical aspects of PCR-based tests.

scholarly journals A Modified SDS – Based Method Applied for Extraction of High-Quality DNA from Raw Corn and Roasted Soybean

2020 ◽  
Vol 43 (1) ◽  
pp. 61-67
Author(s):  
Arita Sabriu-Haxhijaha ◽  
Gordana Ilievska ◽  
Velimir Stojkovski ◽  
Katerina Blagoevska
Keyword(s):  
Solvent Extraction ◽  
Organic Solvent ◽  
Dna Extraction ◽  
High Quality ◽  
High Molecular Weight Dna ◽  
Soybean Sample ◽  
Dna Yield ◽  
Extraction Protocol ◽  
Organic Solvent Extraction ◽  
Corn Kernels

AbstractThe probability of contamination of non-transgenic varieties with genetically modified (GM) products increase as a result of global expansion of areas sown with transgenic crops. DNA-based methods as accurate, efficient and reliable methods are preferable for detection of GM material in raw or highly processed foods. Isolation of high quality DNA with a suitable and efficient DNA extraction protocol is crucial for getting precise results in DNA amplification. In this study, we performed modifications of previously known Sodium dodecyl sulfate (SDS)-based DNA extraction method regarding the incubation period, DNA pellet washing and addition of organic solvent extraction, to improve DNA quality and to reduce costs. Raw corn kernels and roasted soybean seed were used as samples. DNA was extracted following three protocols, modifications of Edwards protocol. The type of detergent used in raw corn sample did not cause significant effects on extracted DNA yield and purity, while in roasted soybean samples the 2% (w/v) SDS lysis buffer gave the highest DNA yield. The additional incubation step raised the DNA yield from raw corn for 121%, while the purest DNA from soybean sample was obtained using organic solvent extraction. Electrophoretic determination of DNA integrity showed varying degree of DNA smearing from roasted soybean. Contrary, all extraction protocols used on raw corn kernels produced a high molecular weight DNA. Thus, our in-house DNA extraction protocol is as efficient but more cost effective compared to commercial kits and can be used for raw corn, while the protocol for roasted soybean needs further improvement.

scholarly journals Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification

2019 ◽  
Vol 2 (2) ◽  
pp. 36
Author(s):  
Lorena G. Caligiuri ◽  
Adolfo E. Sandoval ◽  
Jose C. Miranda ◽  
Felipe A. Pessoa ◽  
María S. Santini ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Internal Control ◽  
Large Scale ◽  
Pcr Amplification ◽  
Proteinase K ◽  
Sand Fly ◽  
Regulatory Function ◽  
Sand Flies ◽  
Lysis Buffer ◽  
Commercial Kits

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.

scholarly journals Optimisation of DNA Extraction from Individual Sand Flies for PCR Amplification

2018 ◽  
Author(s):  
Lorena G. Caligiuri ◽  
Adolfo E. Sandoval ◽  
Jose C. Miranda ◽  
Felipe A. Pessoa ◽  
María S. Santini ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Internal Control ◽  
Large Scale ◽  
Pcr Amplification ◽  
Personal Communication ◽  
Proteinase K ◽  
Sand Fly ◽  
Regulatory Function ◽  
Sand Flies ◽  
Lysis Buffer

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analysing parasite infection in Lutzomyia spp. by PCR [1] and, for this reason, we evaluated various modifications on a previously published protocol ([2] and Acardi personal communication). The most significant variation was the use of a different lysis buffer [3] to which added Ca2+ (buffer TESCa), because this ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site [4]. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene [5,6]. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.

scholarly journals A simple "universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification.

1995 ◽  
Vol 4 (6) ◽  
pp. 368-370 ◽  
Author(s):  
D Goldenberger ◽  
I Perschil ◽  
M Ritzler ◽  
M Altwegg
Keyword(s):  
Dna Extraction ◽  
Extraction Procedure ◽  
Pcr Amplification ◽  
Proteinase K ◽  
Direct Pcr

scholarly journals Comparison of DNA extraction methods for non-marine molluscs: Is modified CTAB DNA extraction method more efficient than DNA extraction kits?

2019 ◽  
Author(s):  
Sudeshna Chakraborty ◽  
Anwesha Saha ◽  
N.A. Aravind
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
High Molecular Weight Dna ◽  
Long Term Storage ◽  
Dna Extraction Method ◽  
Successful Amplification ◽  
Dna Extraction Methods ◽  
Sequence Quality

AbstractIsolation of high molecular weight DNA from gastropod molluscs and its subsequent PCR amplification is considered difficult due to excessive mucopolysaccharides secretion which co-precipitate with DNA and obstruct successful amplification. In an attempt to address this issue, we describe a modified CTAB DNA extraction method that proved to work significantly better with a number of freshwater and terrestrial gastropod taxa. We compared the performance of this method with Qiagen® DNeasy Blood and Tissue Kit. Reproducibility of amplification was verified using a set of taxon-specific primers wherein, modified CTAB extracted DNA could be replicated at least four out of five times but kit extracted DNA could not be replicated. Additionally, sequence quality was significantly better with CTAB extracted DNA. This could be attributed to the removal of polyphenolic compounds by polyvinyl pyrrolidone (PVP) which is the only difference between conventional and modified CTAB DNA extraction methods for animals. The genomic DNA isolated using modified CTAB protocol was of high quality (A260/280 ≥ 1.80) and could be used for downstream reactions even after long term storage (more than two years).

Sign in / Sign up

Export Citation Format

Share Document