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Effects of Thermal Cycling on Surface Hardness, Diametral Tensile Strength and Porosity of an Organically Modified Ceramic (ORMOCER)-Based Visible Light Cure Dental Restorative Resin
Published in P. Mereena Luke, K. R. Dhanya, Didier Rouxel, Nandakumar Kalarikkal, Sabu Thomas, Advanced Studies in Experimental and Clinical Medicine, 2021
The prepared samples were allowed to expose cyclic temperature changes in distilled water from 5°C to 55°C in a dental thermal cycler (Willytec, Germany). The dwell time used was 15 sec at 5°C and 55°C. A 15 sec time was given as the drain time at 22°C. Samples were subjected to 500, 1000, 1500, and 2000 cycles. The thermo cycled samples were used for microCT, microhardness, and DTS evaluation. For microCT evaluation, the same sample before and after thermal cycling was evaluated at each cycles up to 2000 cycles of thermal cycling.
Rapid HLA-DR-Dw and DP Matching by PCR Fingerprinting and Related DNA Heteroduplex Technologies
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
Examine over a UV transilluminator and compare the relative intensities of sample and DPB-UHG PCRs and adjust concentrations to give the same intensity of fluorescence. Add appropriate amounts (6 to 20 μl) of both PCR products to a single microcentrifuge tube, and subject to a further 3 PCR cycles as in ‘Thermal Cycling’. Analyze a 12-μl aliquot for heteroduplexes as described in "Polyacrylamide Minigel Electrophoresis".
100 MCQs from Dr. David Browne and Colleagues
Published in David Browne, Selena Morgan Pillay, Guy Molyneaux, Brenda Wright, Bangaru Raju, Ijaz Hussein, Mohamed Ali Ahmed, Michael Reilly, MCQs for the New MRCPsych Paper A, 2017
Dr Karen Fleming, Dr Michael Kenewali, Dr Manas Sarkar, Dr Daniel White
PCR is a method to selectively amplify small regions of the genome up to 10 000-fold. This eliminates the need to perform a Southern blot analysis. It is a technique to amplify DNA resulting in numerous copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. (31, pp 158–9)
Very early prenatal diagnosis of Cockayne’s syndrome by coelocentesis
Published in Journal of Obstetrics and Gynaecology, 2022
Antonino Giambona, Margherita Vinciguerra, Filippo Leto, Filippo Cassarà, Gaspare Cucinella, Valentina Cigna, Emanuela Orlandi, Maria Piccione, Francesco Picciotto, Aurelio Maggio
PCR was performed in 50 μL reaction using specific primers to amplify the fragment of ERCC6/CSB gene containing molecular defect (Table 1) and a mix of primer to amplify many short tandem repeats (STRs) of highly variable chromosomal markers located on chromosome 13 (D13S325, D13S631, D13S634, D13S305), chromosome 18 (D18S535, D18S386, D18S499, D18S391), chromosome 21 (D21S1435, D21S1411, D21S1442, D21S1414), chromosomes X and Y (AMXY, HPRT, SRY, DXS8377, DXS1187, DXS6803, DXS6809) in order to check eventually maternal DNA contamination. Twenty-five microlitres of 2X Platinum Multiplex PCR Master Mix (Life Technologies, Carlsbad, CA), 10 pm/µL of each forward and reverse primer to amplify ERCC6 gene, 0.3 μL of a mix containing 3–10 pmol of each primer for STR and 11.5 μL of distilled water were added into the tubes containing lysed cells. Maternal and paternal DNA were separately amplified using primers for STR markers. After one cycle of denaturation at 95 °C for 2′, PCR was performed for 25 repeating cycles at 95 °C 30″, 60 °C 90″, 72 °C 60″ and a final cycle at 72 °C for 30′. Thermal cycling was performed using the Gene Ampl 9700 (Applied Biosystems, Foster City, CA).
PreservCyt Is an Optimal Fixative that Permits Cytologic and Molecular Analyses of Vitreoretinal Lymphoma Biopsies
Published in Ocular Immunology and Inflammation, 2021
Mona Meng Wang, Wei Jian Tan, Tong Seng Lim, Anita Sook Yee Chan
MYD88 mutation analysis was performed by PCR. Briefly, final reaction volumes of 50 μl contained 50–100 ng DNA, 10 pmol primers (forward 5ʹ-GTTGTTAACCCTGGGGTTG-3ʹ and reverse 5ʹ-TGCAGGGGTTGGTGTAGT-3ʹ)14, 10 μM dNTP, 1.5 mM MgCl2, 1 U AmpliTaq Gold enzyme (Life Technologies) and ABI Buffer II (Life Technologies). Thermal cycling consisted of: 95°C for 7 min; 35 cycles of 95°C 45 s, 60°C 45 s, 72°C 90 s; 72°C for 10 min. The PCR product was purified on a 1% TAE agarose gel run at 100V, followed by gel extraction using a QIAquick® Gel Extraction Kit (Qiagen) before Sanger sequencing (Bio Basic Asia) for MYD88 mutation(s). IgH gene rearrangement was performed using an IgH Somatic Hypermutation Assay Megakit V2.0 (Invivoscribe) in accordance with the manufacturer’s protocol.
In vitro and ex vivo expression of serum amyloid A3 in mouse lung epithelia
Published in Experimental Lung Research, 2020
Haruka Kawasaki, Tomoaki Murakami, Yassien Badr, Sato Kamiya, Kaori Shimizu, Ayaka Okada, Yasuo Inoshima
RNA extracted from the C22 cells, and lung tissue samples was quantified using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The RNA was stored at −80 °C until required. Contaminating DNA was eliminated by DNase I treatment, and cDNA was synthesized as previously described.20 The synthesized cDNA was used for quantitative real-time PCR analysis. The quantitative real-time PCR procedure and thermal cycling conditions have been described previously.20 We used specific primers for SAA1, SAA3, SP-A, SP-B, SP-C, SP-D, mucin 5AC (MUC5AC), mucin 5B (MUC5B), TNF-α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Supplemental Table S2). mRNA expression was normalized to that of the endogenous gene GAPDH, and fold-changes relative to control levels were determined by the ΔΔCt method.21 All experiments were independently performed at least three times. The collected data are expressed as means ± standard deviations. Statistical significance was determined by Tukey’s post hoc analysis.