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Coat Protein
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
The appropriate isolation techniques were reviewed thoroughly by Weber and Konigsberg (1975). According to this review, the choice of procedure depended on whether it was desired to have the RNA or the protein in native form. One method involved treating the phage solution with urea and LiCl when the RNA precipitated and was removed by centrifugation (Nishihara et al. 1970). The coat protein remained in solution and was used when the native protein was required. Another method, which destroyed the RNA but left the coat protein in solution in a relatively native state, was to treat the phage solution with 50% acetic acid (Fraenkel-Conrat 1957). The RNA precipitated, while the coat protein remained in the supernatant and was acceptable for the phage or capsid reconstitution. The undegraded RNA could be obtained from the phage by phenol extraction when the RNA remained in the aqueous phase and the coat protein precipitated at the interface between the aqueous and phenol layers. The protein could then be useful for structural studies after washing the precipitate with ethanol and ether to remove phenol. Finally, the protein could also be prepared by heating the phage to 100°C for 10 minutes in 2 M KCl (Konigsberg et al. 1966).
A Survey of Newer Gene Probing Techniques
Published in Victor A. Bernstam, Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
Procedures have been developed to yield undegraded nucleic acids from a large number of small samples virtually at the same time, without the use of conventional phenol extraction.
Molecular Methods for the Diagnosis of Fungal Infections
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
Adam M. Bressler, Christine J. Morrison
In another study, a noncommercial disruption and extraction method using lyticase, glass bead beating, and phenol was compared to four commercial kits for the recovery and amplification of Aspergillus fumigatus DNA from bronchoalveolar lavage (BAL) fluid and biopsied tissues.22 The High Pure PCR Template Preparation Kit produced DNA that allowed a limit of sensitivity similar to the lyticase method (10 CFU/reaction), whereas the QIAamp DNA Mini Kit, Masterpure DNA Purification Kit, and DNA-Pure Yeast Genomic Kit were all one to two logs less sensitive than either of the former methods. Furthermore, the addition of a bead-beating step (beating samples with glass beads for 1 min before phenol extraction) improved the sensitivity of the lyticase method by an additional log but did not improve the sensitivity of the High Pure Kit method.
Psychosis susceptibility zinc finger protein 804A (ZNF804A) gene polymorphism in schizophrenia patients treated with olanzapine in North Indian population
Published in International Journal of Neuroscience, 2023
Sayantika Maity, Murali Munisamy, Rajesh Sagar, Nayanabhirama Udupa, Venkata Puluturu Shilpa, Vivekanandhan Subbiah
Three to five mililitres peripheral blood samples, anti-coagulated with EDTA, were collected from all participants with inform consent form. Genomic DNA extraction was performed by standard chloroform-phenol extraction method. Polymerase chain reaction (PCR) amplification of the rs1344706 A > C polymorphism was carried out with the following primers: Forward: 5’- TCAGCCTGAATGGCTTCTCT-3’ and reverse: 5’-AGTGACCTTGGTGGAAATGG-3’. PCR was performed in a total volume of 25 µl, including 25 mmole/L Mgcl2, 10xPCR buffer, 2 mmole/L dNTP, 25 pmole of each primer, 1 U Taq DNA polymerase and 2 µg genomic DNA, using a thermal cycler (Eppendorf) by initial denaturation 940c for 30 s, annealing at 600c for 40 s and extension at 72 °C for 40 s, followed by a final extension at 72 °C for 7 min (Figure 1). After PCR, genotyping was performed by SNaPshot methods (Applied Biosystems) for all the patients and controls who participated in this study.
Effects of gamma irradiation on morphology and protein differential in M1V1 population of Vanilla planifolia Andrews
Published in International Journal of Radiation Biology, 2023
Rohayu Ma’Arup, Nur Syazwani Ali, Fisal Ahmad, Zaiton Ahmad, Mohamad Feisal Mohamed Norawi, Homaa Faezah Moinuddin
Phenol extraction methanol-ammonium acetate precipitation method was performed (Carpentier et al. 2005, Rodrigues et al. 2009; Azwan et al. 2010) with some modifications. 10 mL of extraction buffer (50 mM Tris-HCL pH 8.5, 100 mM KCl, 1% v/v DTT, and 30% w/v sucrose) were added to the sample mixture. The sample was vortexed for 15 min and centrifuged 10,000 × g for 3 min at 4 °C. The phenolic phases were collected and re-extracted with an equal volume of extraction buffer. The phenolic phases were left to precipitate overnight with five times volume of cooled 100 mM ammonium acetate in methanol at −20 °C. The supernatant was discarded after centrifuge (10,000 × g, 30 min at 4 °C). The resulting pellet was washed twice with ice-cold acetone containing 0.2% v/v DTT and between two rinsing steps; the sample mixture was left for 1 h at −20 °C. The pellet was vortexed for 10 min at 4 °C during each wash to remove other interfering compounds. The pellet was air-dried at room temperature and the pellet was re-suspended in 300 µL solubilization buffer containing 7 M urea, 2 M thiourea, 4% v/v CHAPS, 0.2% v/v Bio-Lyte 3–10 and 1% v/v DTT. The sample mixture was vortexed every 15 min for 1 h at room temperature. The supernatant was collected after centrifuge and stored at −80 °C in an airtight container until further analysis.
Recent advances in lipopolysaccharide-based glycoconjugate vaccines
Published in Expert Review of Vaccines, 2021
Henderson Zhu, Christine S. Rollier, Andrew J. Pollard
Refinements to the hot phenol-water method were made over time to address several aims: to adapt to different scales of operation, to improve yield, and to improve purity [56,57]. Notably, the initial drawback of the hot phenol-water method is the enrichment of contaminants in the preparation, including RNA, DNA, and lipoproteins. These contaminants are difficult to isolate and can confound downstream applications relying on the biological properties of the LPS, such as the production and validation of glycoconjugates. To minimize these contaminants, enzymatic pre-treatments with RNase A, DNase I, and proteinase K before the phenol-water extraction are now standard [58–60]. To achieve an even higher purity of LPS with the aim of removal of remaining lipoproteins, repurification methods has been developed using phenol extraction [61].