Abi prism 3130xl

Genomic DNA was extracted from the legs of each pupa (right leg of the second pair) or from ca. $1/3-1/2$ of the larvae body using DNeasy Blood and Tissue Kit (Qiagen GmbH, Hilden, Germany) according to manufacturer’s instructions.
The PCR primers used were those developed for P. dominula (Henshaw 2000 (link)). PCRs were carried out in 5 μl reaction volumes containing 1× Type-it Microsatellite Kit (Qiagen), 0.07 μM forward primer, 0.2 μM reverse primer, 0.14 μM fluorescent-labeled M13 primer, and 1 μl (6–65 ng) of DNA template using a thermocycling profile of one cycle of 5 min at 96 °C followed by 30 steps of 30 s at 95 °C, 90 s at 50 °C, and 30 s at 72 °C, with a final step of 30 min at 65 °C.
Amplicons were fluorescently labeled in multiplexed reactions using a modified M13-tailing method (Oetting et al. 1995 (link)). The amplified alleles were separated on an ABI PRISM 3130XL (Applied Biosystems) with Genescan 600LIZ size standard and scored with the program Peak Scanner v 1.0 (Applied Biosystems).

All blood samples from howler monkeys were screened for the presence of Mansonella and Brugia DNA using the genus specific PCR. The PCR reactions were carried out in a total volume of 50 µL, comprising 25 µL of AmpliTaq Gold master mix (Thermo Fisher Scientific, Saint Herblain, France), 18 µL of ultrapure water free of DNAse-RNAse, 1 µL of each primer and 5 µL of genomic DNA. PCR reactions were run under the following protocol: the incubation step at 95 °C for 15 min, 40 cycles of one minute at 95 °C, 30 s for the annealing at a different melting temperature for each PCR assays (Table 3), and 72 °C of elongation step (Table 3) with a final extension step of five minutes at 72 °C. PCR reactions were performed in a Peltier PTC-200 model thermal cycler (MJ Research Inc., Watertown, MA, USA).
DNA amplicons generated throughout each PCR reaction were purified using NucleoFast^® 96 PCR DNA purification plate (Macherey Nagel EURL, Hoerdt, France). Purified DNAs were subjected to the second amplification using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Perkin Elmer Applied Biosystems, Foster City, CA, USA), then the BigDye PCR products were purified on the Sephadex G-50 Superfine gel filtration resin prior to sequencing on the ABI Prism 3130XL (Applied Biosystems, Courtaboeuf, France).

DNA was extracted from the blood leukocyte fraction using the phenol-chloroform method. Amplification of non-coding (exon 1), coding (exon 2) and flanking intronic regions of the GJB2 gene was conducted with PCR on a MJ Mini (Bio-Rad) thermocycler using primers 5'-CCGGGAAGCTCTGAGGAC-3' and 5'-GCAACCGCTCTGGGTCTC-3' for amplification of exon 1 [55 (link)] and 5'-TCGGCCCCAGTGGTACAG-3' and 5'-CTGGGCAATGCGTTAAACTGG-3' for amplification of exon 2 [32 (link), 58 (link), 59 (link)]. The PCR products were subjected to direct sequencing using the same primers on ABI PRISM 3130XL (Applied Biosystems, USA) Genomics Core Facility, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia). DNA sequences variations were identified through comparison with the GJB2 gene reference sequences М86849.2 and U43932.1 (GenBank).

Eighteen HPIV‐3‐positive specimens were randomly selected and sequenced. Reverse transcription and amplification of HN gene were carried out using the One‐Step RT‐PCR Kit (Qiagen) with primers described previously.8 PCR products were selected and purified using EZNA Gel Extraction Kit (Omega, Norcross, GA, USA) and then sequenced by ABI Prism 3130xl automated sequencer (Applied Biosystems, Foster City, CA, USA) (Supporting information 3).

Total mRNA from the patient’s fibroblasts was obtained using TRIzol reagent (Life Technologies) and cDNA was synthesized using M-MLV Reverse Transcriptase (Life Technologies). The cDNA-based PCR product corresponding to the coding sequence of ALG1 was obtained using forward primer ALG1s 5′-TGACTGCTGCGGGCCAG-3′ and reverse primer ALG1as 5′-CACTGGGAGGTGCTGCTCG-3′. In the case of the patient, the amplicon was isolated in low melting point agarose gel, purified, subcloned, and screened for alternative splicing.
The inheritance of variants was determined by analyzing the patient’s and parents’ gDNA using primer ALG1s and reverse primer ALG1gas 5′-CTAAAGGAGCACTTCCGCC-3′ for the c.208 + 25G > T pathogenic variant and forward primer ALG1-E13s 5′-CAGGCAATGAGGTAAGCTCTG-3′ and reverse primer ALG1-E13as 5′-CAATTCTTTTACCAGGCAGTACC-3′ for the c.1312C > T pathogenic variant. Sequencing was performed by an ABI Prism 3130xl autoanalyzer (Applied Biosystems, Foster City, CA, United States), and results were visualized using SnapGene Viewer 2.2.2 (GSL Biotech LLC, Chicago, IL, United States).