Prestaining of PCR products with SYBR Green for agarose gel electrophoresis: advantages and limitations

2011 ◽  
Vol 49 (6) ◽  
Author(s):  
Robert Konschak ◽  
Ingeborg Tinhofer
Keyword(s):  
Gel Electrophoresis ◽  
Sybr Green ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Pcr Products

scholarly journals SEX DETERMINATION OF BALI STARLING (Leucopsar rothschildi) USING MOLECULAR SEXING

2015 ◽  
Vol 2 (1) ◽  
pp. 114
Author(s):  
Putu Indra Pramana Wirastika ◽  
Ignatius Pramana Yuda ◽  
Felicia Zahida
Keyword(s):  
Dna Binding ◽  
Sex Determination ◽  
Gel Electrophoresis ◽  
Morphological Characters ◽  
Molecular Sexing ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Pcr Products ◽  
Conventional Methods

<p>Bali Starling (Leucopsar rothschildi) are monomorphic at the age of nestling. For the conservation of bird it important is to determine its sex at the earlier stage. Conventional methods have limitations. This study applied PCR-based molecular sexing to answer this issue. This study aimed to obtain the most effective molecular primers to identify the sex of Bali starling. The most common used combination of P2/P8, 2550F/ 2718R and 1237L/1272H primers, which amplify CHD1 gene (Chromo-helicase-DNA-binding) were evaluated. DNA samples were obtained from secondary wing feathers of young Bali Starling. Separation in agarose gel electrophoresis of PCR products showed that the three primers were successfully amplified the samples with different degrees of success, that was 90% (P2/P8), 86.7% (2550F/2718R), and 73.3% (1237L/1272H), respectively. However, only the combination of P2/P4 and 2550F/2718R primers was able to sex Bali Starling based on observation of PCR products on agarose gel. The sizes of the genes were slightly different with those reported on previous studies. Most of the results of molecular sexing were in accordance with the sex based on morphological characters.</p><p><br /><strong>Keywords</strong> : Bali starling, Leucopsar rothschildi, molecular sexing, CHD gene</p>

scholarly journals Genetic variability of broodstocks of restocking programs in Brazil

2015 ◽  
pp. 4677-4687 ◽  
Author(s):  
Nelson Lopera-Barrero ◽  
Ed Lima ◽  
Luiz Filho ◽  
Elenice Goes ◽  
Pedro Castro ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Genetic Variability ◽  
Gel Electrophoresis ◽  
Shannon Index ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
General Management ◽  
Rhamdia Quelen ◽  
Pcr Products ◽  
Salminus Brasiliensis

ABSTRACT Objective. The aim of this study was evaluate the genetic diversity of the following broodstocks: piapara (Leporinus elongatus), dourado (Salminus brasiliensis), jundiá (Rhamdia quelen) and cachara (Pseudoplatystoma fasciatum) already useful for restocking programs in the Paranapanema, Iguaçu and Paraná Brazilian Rivers. Materials and methods. Samples from the caudal fin of 122 fish were analyzed. DNA was extracted by NaCl protocol. PCR products were separated by a horizontal agarose gel electrophoresis. The fragments were visualized by staining with ethidium bromide. Results. The amplification of 25 primers generated different fragments in studied species that allowed characterizing 440 fragments of 100-2900 bp. High percentage of polymorphic fragments (66.67 to 86.29), Shannon index (0.365 to 0.486) and genetic diversity of Nei (0.248 to 0.331) were detected. Conclusions. The level of genetic variability in the broodstocks was adequate for allowing their use in restocking programs in the studied Rivers. However, periodical monitoring studies of genetic variability in these stocks, the mating system, reproductive system and general management must be made to guarantee the preservation of wild populations.RESUMEN Objetivo. El objetivo de este estudio fue evaluar la diversidad genética de los siguientes lotes de reproductores: piapara (Leporinus elongatus), dourado (Salminus brasiliensis), jundiá (Rhamdia quelen) y cachara (Pseudoplatystoma fasciatum) utilizados para programas de repoblación en los ríos brasileños Paranapanema, Iguaçu y Paraná. Materiales y métodos. Muestras de aleta caudal de 122 peces fueron analizadas. El ADN fue extraído por el protocolo de NaCl. Los productos de PCR fueron separados por electroforesis horizontal en gel de agarosa. Los fragmentos fueron visualizados por marcación con bromuro de etidio. Resultados. La amplificación de los 25 iniciadores produjo diferentes fragmentos en las especies estudiadas que permitieron caracterizar 440 fragmentos de 100 a 2900 pb. Fueron detectados un alto porcentaje de fragmentos polimórficos (66.67 a 86.29), de índice de Shannon (0.365 a 0.486) y de diversidad genética de Nei (0.248 a 0.331). Conclusiones. El nivel de variabilidad genética en los lotes de reproductores fue adecuado para su utilización en programas de repoblación en los ríos estudiados. Sin embargo, estudios de monitoreo periódico de la variabilidad genética en esos lotes, del sistema de cruzamiento, del sistema reproductivo y del manejo general deben ser realizados para garantizar la preservación de las populaciones naturales. 

High-density fluorescence in situ hybridization signal detection on barley (Hordeum vulgare L.) chromosomes with improved probe screening and reprobing procedures

Genome ◽  
2011 ◽  
Vol 54 (2) ◽  
pp. 151-159 ◽  
Author(s):  
Akio Kato
Keyword(s):  
In Situ Hybridization ◽  
Hordeum Vulgare ◽  
Fluorescence In Situ Hybridization ◽  
Gel Electrophoresis ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Hordeum Vulgare L ◽  
Pcr Products ◽  
Break Points

The barley ( Hordeum vulgare L.) genome was screened to identify sequences that could be used for fluorescence in situ hybridization (FISH). From 2000 transformed bacterium colonies carrying barley clones, 56 colonies were selected on the basis of the patterns that their PCR products produced when subjected to agarose gel electrophoresis. Among them, 42 (75%) exhibited fluorescent signals on barley chromosomes after in situ hybridization using the directly labeled PCR products. Sequencing revealed seven clones, pHv-365, pHv-177, pHv-1112, pHv-689, pHv-1476, pHv-1889, and pHv-1972, to be newly identified FISH-positive sequences. The remainder possess previously described sequences such as 5S, GAA microsatellite, centromere repeats, HVT01, and pHvMWG2315 (324 bp repeat). It is shown here that a combination of five probes, which produce strong signals on barley chromosomes, pHv-38 (5S), pHv-365, pHv-961 (HVT01), GAA, and TAG microsatellites, offer unequivocal recognition of each chromosome. The combination of three probes, i.e., pHv-1123 (barley 324 bp repeat), GAA, and TAG, decorated entire chromosomes with fine dotted signals and was useful for detecting the break points of aberrant chromosomes. The signals’ distributions of pHv-177, pHv-1112, and TAG were highly polymorphic. An improved reprobing procedure and its usefulness are also discussed.

scholarly journals The optimal condition of agarose gel electrophoresis to visualize the PCR products obtained from AMPLICOR CMV PCR test.

1998 ◽  
Vol 42 (3) ◽  
pp. 181-184
Author(s):  
Shigemi Hashimoto ◽  
Motofumi Hiyoshi ◽  
Takayuki Takubo ◽  
Shinichi Tagawa ◽  
Noriyuki Tatsumi
Keyword(s):  
Optimal Condition ◽  
Gel Electrophoresis ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Pcr Products ◽  
Pcr Test

scholarly journals Detection of Acanthamoeba spp. in two major water reservoirs in the Philippines

2020 ◽  
Vol 18 (2) ◽  
pp. 118-126 ◽  
Author(s):  
Giovanni Milanez ◽  
Frederick Masangkay ◽  
Frieda Hapan ◽  
Thea Bencito ◽  
Marcus Lopez ◽  
...  
Keyword(s):  
Water Samples ◽  
Gel Electrophoresis ◽  
Water Security ◽  
The Philippines ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Water Reservoirs ◽  
Chain Reaction ◽  
Pcr Products ◽  
Polymerase Chain

Abstract Water reservoirs are important manmade structures providing water security to deliver clean and safe water for drinking and other purposes to the community. Eighty water samples were collected from Magat and Ipo water reservoirs using purposive sampling between November 2018 and January 2019. Water samples were collected in sterile containers for testing. The samples were cultured in non-nutrient agar and lawned with Escherichia coli and incubated at 33 °C. Twelve out of the 80 (15%) water samples were positive for amoebic growth. Light and scanning electron microscopy (SEM) revealed double-walled cystic stages and were initially identified as Acanthamoeba spp. based on morphological characteristic in reference to Page's established criteria. Their extracted DNAs were used in polymerase chain reaction using JDP1 and JDP2 primers and confirmed the presence of Acanthamoeba DNA in agarose gel electrophoresis. Aligned sequences from PCR products were deposited in GenBank under accession numbers MK886460, MK909919, MK905437, MK910997, MK911021 and MK886514. The presence of potentially pathogenic Acanthamoeba spp. in water reservoirs is considered a potential risk for public health, requiring appropriate processing of water in treatment plants.

scholarly journals Prevalence of CYP2C19 and ITGB3 polymorphisms among Bangladeshi patients who underwent percutaneous coronary intervention

2021 ◽  
Vol 9 ◽  
pp. 205031212110422
Author(s):  
Md Rabiul Islam ◽  
Tasnova Tasnim Nova ◽  
NAM Momenuzzaman ◽  
Sikder Nahidul Islam Rabbi ◽  
Ishrat Jahan ◽  
...  
Keyword(s):  
Percutaneous Coronary Intervention ◽  
Genetic Polymorphisms ◽  
Gel Electrophoresis ◽  
Coronary Intervention ◽  
Amplification Refractory Mutation System ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Arms Pcr ◽  
Percutaneous Coronary ◽  
Pcr Products

Introduction: Antithrombotic agents are the basic therapeutic option for patients with arterial thrombosis who underwent percutaneous coronary intervention (PCI). In Bangladesh, aspirin and clopidogrel are frequently prescribed as antithrombotics or platelet inhibitors. Studies reported the genetic polymorphisms of CYP2C19*2, CYP2C19*17, and ITGB3 cause an alteration of the pharmacodynamic and pharmacokinetic profile of aspirin and clopidogrel. Therefore, we aimed to assess the prevalence of CYP2C19*2, CYP2C19*17, and ITGB3 polymorphisms among Bangladeshi patients with cardiovascular disease (CVD) who underwent PCI. Methods: Here we assessed a total of 1,000 CVD patients (male 782 and female 218) who underwent PCI and were treated with clopidogrel and/or aspirin. We performed genotyping of patients treated with clopidogrel and aspirin by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) and tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) methods. The PCR products of clopidogrel-treated patients were screened with agarose gel electrophoresis and then digested with SmaI and NsiI-HF for CYP2C19*2 and CYP2C19*17, respectively. We genotyped aspirin-treated patients with T-ARMS-PCR for missense rs5918 (PlA1/A1) polymorphism of the ITGB3 gene. Then we ran the digested PCR products on 2% agarose gel electrophoresis to detect the mentioned polymorphisms. Results: Among the clopidogrel-treated patients, we observed 64.1% polymorphism (hetero + mutant) of CYP2C19*2 (loss-of-function allele) and 22.7% (hetero + mutant) of CYP2C19*17 (gain-of-function allele). On the other hand, among the aspirin-treated patients, polymorphisms of ITGB3 were 84.1% homozygous (PlA1/A1), 15.6% heterozygous (PlA1/A2), and 0.3% mutant homozygous. Conclusion: In the present study, we observed a high prevalence of genetic polymorphisms of CYP2C19 and ITGB3 genes. Therefore, we recommend genotyping of CVD patients before prescribing clopidogrel or aspirin to prevent coagulation. Based on the genotyping study, the adjustment of doses or alternative generics might require to avoid therapeutic failure or toxicity in some cases.

Luminography – an Alternative Assay for Detection of von Willebrand Factor Multimers

1988 ◽  
Vol 60 (02) ◽  
pp. 133-136 ◽  
Author(s):  
R Schneppenheim ◽  
H Plendl ◽  
U Budde
Keyword(s):  
Von Willebrand Factor ◽  
Gel Electrophoresis ◽  
Detection Methods ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Luminescence Assay ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA luminescence assay was adapted for detection of von Willebrand factor multimers subsequent to SDS-agarose gel electrophoresis and electroblotting onto nitrocellulose. The method is as fast as chromogenic detection methods and appears to be as sensitive as autoradiography without the disadvantages of the latter.

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