General Taq PCR Master Mix -- CHEM 384/584 v2

2X PCR Master Mixes are convenient to use as they include all the necessary PCR components except the template and primers. Most PCR Master Mixes also include agarose gel running dyes and a density reagent that allows direct loading of PCR products on an agarose gel for electrophoresis. The master mix format simplifies workflows and sample handling; simply add primers, template, and water and then begin PCR.

Investigation of the Effect of Agarose Gel Concentration and Culture Period on Bio and Mechanical Properties of Chondrocyte Tissue Engineering

As a gel scaffold for chondrocyte tissue engineering, agarose concentration plays a significant role in the relationship between porosity and nutrition. In this work, the effect of concentration and period cultured on Glycosaminoglycan (GAG) and mechanical properties have been studied. A bovine chondrocytes have been isolated and seeded in different agarose gel scoffed concentrations, about 4% and 6%, for different period cultured, 0 and 7 days. The MTS machine and Spectrophotometric with calibration curve method were used to measure mechanical properties, and GAG concentration of the prepared samples, respectively. The results of mechanical tests and GAG contents shown that there are a wide range of dispersion in the most of the samples, which attribute to different factors. For mechanical properties, these factors could be attributed to anisotropic of the produced chondrocyte with agarose scaffolds, insufficient cells' dispersion within the gel scaffold during seeding and cultured time, and some test procedure condition, such as EBSS hydration. While for GAG results, those factors could be the differences of the cell growth environment between in-vitro and in vivo media.

Frequencies of CYP2B6∗4,∗5, and ∗6 Alleles within an Iranian Population (Mazandaran)

Background. The human CYP2B subfamily consists of one functional gene (CYP2B6) and one pseudogene (CYP2B7P). Cytochrome P450 2B6 (CYP2B6) is a highly polymorphic enzyme that shows marked interindividual and interethnic variations. Currently, 38 alleles have been described, and some of the allelic variants have been associated with low enzyme activity. The aim of this study was to investigate the frequencies of CYP2B6∗4, CYP2B6∗5, and CYP2B6∗6 alleles in the Mazani ethnic group among Iranian Population. Methods. The study was conducted in 289 unrelated healthy volunteers. DNA was extracted from peripheral blood and analyzed by the PCR-RFLP protocol. The PCR product was digested with restriction enzymes and then separated using agarose gel electrophoresis. Results. The frequency of CYP2B6∗4, CYP2B6∗5, and CYP2B6∗6 in this study was 34.60%, 7.26%, and 34.54%, respectively. Conclusion. The frequency of the CYP2B6∗4 allele in the Mazani ethnic group was much higher (34.60%) than other population. The frequency of CYP2B6∗6 (34.54%) also was higher than its frequency in other previously reported population. But the frequency of CYP2B6∗5 in this study was lower than expected. These results will be useful in understanding the ethnic diversity in Iranian population and offer a preliminary basis for more rational use of drugs that are substrates for CYP2B6 in this population.

Low-Cost Imaging of Fluorescent DNA in Agarose Gel Electrophoresis Using Raspberry Pi Cameras

Low-cost analytical solutions built around microcomputers like the Raspberry Pi help to facilitate laboratory investigations in resource limited venues. Here, three camera modules (V1.3 with and without filter, as well as NoIR) that work with this microcomputer were assessed for their suitability in imaging fluorescent DNA following agarose gel electrophoresis. Evaluation of their utility was based on signal-to-noise (SNR) and noise variance metrics that were developed. Experiments conducted with samples were subjected to Polymerase Chain Reaction (PCR), and the amplified products were separated using gel electrophoresis and stained with Midori green. Image analysis revealed the NoIR camera performed the best with SNR and noise variance values of 21.7 and 0.222 respectively. In experiments conducted using UV LED lighting to simulate ethidium bromide (EtBr) excitation, the NoIR and V1.3 with filter removed cameras showed comparable SNR values.

Molecular Classification of Vicia faba L Genotypes by Using RAPD-PCR Markers

The research included the molecular classification study of seven genotypes of the bean Vicia faba L. (FBSPN2, TLD1266, TLD1814, TLB1266, Luzdeotono, favad and Histal. Using the RAPD technique for DNA, as 13 random primers were used, the products of inflation were transferred within the agarose gel, and the results of the study showed the possibility of separating the genotypes from each other and determining the degree of genetic variation between them, as the primers used produced (1002) packages of them (417 normal bundles and (585) mixed bundles. The genetic differences of the studied genotypes were determined to be distinguished by the number of bundles, as they reached (28) bundles, including (13) unique bundles and (15) absent bundles. The ILB1266 genotype showed the highest number of unique bundles, which It reached 4 bundles, while the cultivar Favad showed the absence of unique bundles in it, either bundles are absent. The genotypes (ILD1266, IILB1266, Luzdeotono) were distinguished for having the highest number, which amounted to (3) bundles. As for the FBSPN2 genotype, it did not have any absent bundle, and the primers varied. Of the resulting bundle sizes, their sizes ranged between bp (1925-130), and the highest value for the genetic dimension ranged between (0.110 - 0.269), as the lowest genetic dimension was between the two structures (FBSPN2 and ILD1266), which amounted to 0.110, and the highest value for the genetic dimension was (0.2 69) between the genotypes (ILD1266, HISTAL) (ILD1266, Luzdeotono) The Dendrogram shows the separation of the studied genotypes into two main groups, and each of them into two subgroups.

High sensitivity-low cost detection of SARS-CoV-2 by two steps end point RT-PCR with agarose gel electrophoresis visualization

More than one year since Coronavirus disease 2019 (COVID-19) pandemic outbreak, the gold standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is still the RT-qPCR. This is a limitation to increase testing capacities, particularly at developing countries, as expensive reagents and equipment are required. We developed a two steps end point RT-PCR reaction with SARS-CoV-2 Nucleocapsid (N) gene and Ribonuclease P (RNase P) specific primers where viral amplicons were verified by agarose gel electrophoresis. We carried out a clinical performance and analytical sensitivity evaluation for this two-steps end point RT-PCR method with 242 nasopharyngeal samples using the CDC RT-qPCR protocol as a gold standard technique. With a specificity of 95.8%, a sensitivity of 95.1%, and a limit of detection of 20 viral RNA copies/uL, this two steps end point RT-PCR assay is an affordable and reliable method for SARS-CoV-2 detection. This protocol would allow to extend COVID-19 diagnosis to basic molecular biology laboratories with a potential positive impact in surveillance programs at developing countries.

Alkaline Agarose Gel Electrophoresis

Alkaline agarose gels are run at high pH, which causes each thymine and guanine residue to lose a proton and thus prevents the formation of hydrogen bonds with their adenine and cytosine partners. The denatured DNA is maintained in a single-stranded state and migrates through an alkaline agarose gel as a function of its size. Other denaturants such as formamide and urea do not work well because they cause the agarose to become rubbery.

A Novel Lateral Flow Assay for Rapid and Sensitive Nucleic Acid Detection of Avibacterium paragallinarum

Avibacterium paragallinarum, the pathogen of infectious coryza, caused a highly contagious respiratory disease that poses a serious threat to chickens. Hence, it is necessary to do diagnostic screening for Av. paragallinarum. Existing technologies have been used for Av. paragallinarum testing, which, however, have some drawbacks such as time consuming and expensive that require well-trained personnel and sophisticated infrastructure, especially when they are limitedly feasible in some places for lack of resources. Nucleic acid hybridization-based lateral flow assay (LFA) is capable of dealing with these drawbacks, which is attributed to the advantages, such low cost, rapid, and simple. However, nucleic acid determination of Av. paragallinarum through LFA method has not been reported so far. In this study, we developed a novel LFA method that employed gold nanoparticle probes to detect amplified Av. paragallinarum dsDNA. Compared with agarose gel electrophoresis, this LFA strip was inexpensive, simple- to- use, and time- saving, which displayed the visual results within 5–8 min. This LFA strip had higher sensitivity that achieved the detection limit of 101 CFU/ml compared with 102 CFU/ml in agarose gel electrophoresis. Besides, great sensitivity was also shown in the LFA strip, and no cross reaction existed for other bacteria. Furthermore, Av. paragallinarum in clinical chickens with infectious coryza were perfectly detected by our established LFA strip. Our study is the first to develop the LFA integrated with amplification and sample preparation techniques for better nucleic acid detection of Av. paragallinarum, which holds great potential for rapid, accurate, and on-site determination methods for early diagnosis of Av. paragallinarum to control further spreading.