Nucleofast 96 pcr kit

The DNA amplification was performed on GeneAmp PCR Systems 9700 (Applied Biosystems, Darmstadt, Germany) using a standardized touchdown PCR profile with HotStarTaq DNA Polymerase (Qiagen, Hilden, Germany): initial denaturation for 15 min at 95°C, followed by four cycles of denaturation at 95°C/30 s, annealing at 62°C/30 s (decreasing by 1°C per cycle), extension at 72°C/60 s); then 35 cycles denaturation at 95°C/30 s, annealing at 58°C/30 s, extension at 72°C/60 s; followed by a final extension step at 72°C/7 min. Based on amplicon length, the extension time was modified (1 min/1 kb). The PCR products were resolved by 1.5–2.5% gel-electrophoresis depending on amplicon size. PCR products were purified using the NucleoFast 96 PCR Kit (Macherey-Nagel, Germany) and sequenced using BigDye Terminator chemistry (BigDye^® Terminator v3.1, Applied Biosystems, Darmstadt, Germany) on the 3730xl DNA Analyzer (Applied Biosystems, Carlsbad, CA, United States). Sequence analysis was performed using ‘Sequencher 4’ software (Genecodes Corporation, United States). The identified SNPs between resistant and susceptible genotypes were converted into CAPS markers according to procedures previously described in section marker development and primer design.

Blood samples were incubated in EDTA. Genomic DNA was extracted from peripheral blood leukocytes using the NucleoSpin DNA kit (Macherey-Nagel, Germany). PCR was performed to amplify the relevant gene region (SuperHot Master Mix, Bioron, Germany). The Ugrp2 gene has three exons; therefore, three primer pairs were used for the PCR reaction. For the first exon, 5′-GGTCAGACCGCAAAGCGAAGG-3′ was used as the forward primer and 5′-GACCTGGGATCCACGATCGG-3′ was used as the reverse primer; a 465-bp fragment was amplified. For the second exon, 5′-TGCACAGAGTTCACCGGTCCTTC-3′ was used as the forward primer and 5′-AGGGGCAGGACGGGAAACAG-3′ was used as the reverse primer; a 611-bp fragment was amplified. For the third exon, 5′-CCGCTCCCGCTCCCCACAGA-3′ was used as the forward primer and 5′-TCTCTCCCTCTCTCACGCAGCAC-3′ was used as the reverse primer; a 349-bp fragment was amplified. The NucleoFast 96 PCR kit was used for purification of the amplified products (Macherey-Nagel). Sequencing was performed for Ugrp2. The sequencing reaction was performed using purified PCR products. The obtained sequence products were purified using the ZR Sequencing Clean-up Kit D4051 (Zymo Research, USA) and prepared for array analysis. Polymorphisms in Ugrp2 were identified using the ABI PRISM 3130 Genetic Analyzer capillary automatic sequencing equipment.

To identify Cryptosporidium species/genotypes, secondary PCR products were purified using the NucleoFast^® 96 PCR kit (Macherey Nagel, GmbH & Co KG, Düren, Germany). Purified PCR products were sequenced in both directions, using the secondary PCR primers (Genoscreen, Pasteur Institute of Lille, Lille, France). Obtained nucleotide sequences were aligned using the BioEdit v. 7.0.1 package, and compared with available DNA sequences of Cryptosporidium in GenBank data base using the NCBI BLAST basic local alignment search tool (http://www.ncbi.nlm.nih.gov/BLAST/).
For longer 18S rDNA and actin amplicons, secondary PCR products were purified using a filter tip method [23 (link)] and sequenced in both directions using an ABI Prism™ Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions at 58 °C. Nucleotide sequences identified in this study were deposited in GenBank under the accession numbers: MT570027—MT570035 (actin locus) and MT776545—MT776547 (18S rDNA locus).

To identify Cryptosporidium species or genotypes, positive secondary nested PCR products were purified using the NucleoFast^® 96 PCR kit (Macherey Nagel, GmbH & Co KG, Germany). Purified PCR products were sequenced directly in both directions, using the secondary PCR primers (Genoscreen, Pasteur Institute of Lille, France). Obtained nucleotide sequences were aligned using the BioEdit v7.0.1 package, and compared with available DNA sequences of Cryptosporidium in GenBank data base using the NCBI BLAST basic local alignment search tool¹. Subtyping of C. parvum was based on sequence analysis of the 60 kDa glycoprotein (gp60) gene as previously reported (Gatei et al., 2007 (link)). The amplified DNA fragments were purified, sequenced, and analyzed as described above. All of the nucleotide sequences identified in this study were deposited in GenBank under the accession numbers MK236538-MK236548.

All the PCR products were purified for the NGS processing with the NucleoFast 96 PCR kit (Macherey-Nagel, Dueren, Germany). The purified product was quantitated by spectrophotometry using Nanodrop N1000 (Thermo Fisher, Wilmington, USA). Nucleic acids with concentrations adjusted to 0.2 ng/ul were sequenced on the Nextera XT (Illumina, CA, USA) sequencing platform.
The spike glycoprotein region located between 21709 and 23193 bps in the SARS-CoV-2 genome was aligned with the SARS-CoV-2 Wuhan Hu-1 isolate (GenBank accession number; MN908947.3). The primer pairs R: 5′-acacctgtgcctgttaaaacca-3′ and F: 5′-gacaaagttttcagatcctcagttttaca-3′. were used for sequencing [12 (link)], and sequencing was performed between the 118F–1652R primer region (∼1500 bp) on the Miseq sequencing platform (Illumina, CA, USA). Based on BWA software, all sequencing data were reassigned with Miseq Reporter (https://bio-bwa.sourceforge.net/). The protocol for NGS PCR sequencing was generated as follows: at 45°C for 10 min, at 95°C for 2 min, then for 40 cycles; 95°C for 10 s, 57°C for the 30 s, and 72°C for 30 s.