Illustra gfx pcr dna and gel band purification kit

Routine DNA manipulations were performed as described by Sambrook et al. [21 ]. All restriction enzymes were purchased from Thermo Fisher Scientific and used according to the manufacturers’ recommendations. PCR amplifications were carried out using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). DNA from agarose gels and PCR products were purified with the illustra™ GFX™ PCR DNA and Gel Band Purification kit (GE Healthcare Life Sciences). All DNA ligations were performed using T4 DNA Ligase (Thermo Fisher Scientific). DNA phosphorylation was performed using T4 Polynucleotide Kinase (Thermo Fisher Scientific) and DNA dephosphorylation with FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific). Plasmids were purified using the NZYMiniprep kit (NZYTech). DNA sequencing was performed with the ABI PRISM BigDye^® Terminator Cycle Sequencing Kit (Applied Biosystems). The sequencing reaction was purified by gel filtration and resolved in an ABI 3730XL sequencer.

Sequencing of the 5’ and 3’ termini of the viral genome was performed using a 5’/3’ RACE kit (Roche) following the manufacturer’s protocol. All primers used are described in Table 2. To obtain the 5’ end sequence of the ZIKV genome 5’ RACE was performed. Briefly, 1 μg total RNA was extracted from ZIKV-infected Vero E6 cells using a Direct-zol RNA mini kit and reverse transcribed using the ZIKV specific primer, SP1. The synthesized cDNA was purified using the illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare) according to the manufacturer’s instructions. This was prior to polyadenylation at the 3’ end and amplification using the PCR anchor primer and a ZIKV specific primer (5’ PCR). 3’ RACE was carried out to obtain the 3’ end sequence using 1 μg total RNA extracted from ZIKV infected Vero cells which was polyadenylated at the 3’ end using Poly(A) polymerase (New England Biolabs) following the manufacturer’s guidelines. cDNA synthesis was performed by reverse transcribing the RNA using the oligo (dT) anchor primer. Amplification of the cDNA was achieved by using the PCR anchor primer and a ZIKV specific primer (3’ PCR). The PCR cycling conditions were 95°C for 2 min then 35 cycles of 95°C 20 sec, 56°C (5’ RACE) or 68°C (3’ RACE) for 10 sec, 70°C for 15 sec and 70°C for 7 min.

Genomic DNA was extracted using QIAamp® DNA Mini Kit (Qiagen) and the extracted DNA was bisulfite treated using EZ DNA Methylation-Lightning Kit (Zymo Research). The bisulfite converted p53 promoter region R2, R2-3 and R3 were amplified with the primer sets: F (R2) 5′-GTGTTTTTTTTTTTTTTTGGGAGTAGGTAGAAG-3′, R (R2) 5′-AACCTAAAAAATAAAATACAAAAAAAATACAAAACCTACT-3′, F (R2-3) 5′-GTAGGTTTTGTATTTTTTTGTATTTTATTTTTTAGG-3′, R (R2-3) 5′-CTCATCAATTAAAATATCATTTTTTAAAAAAACTTTCC-3′,F (R3) 5′-TTAATTGATGAGAAGAAAGGATTTAGTTGAGAG-3′, R (R3) 5′-TCATCAAATTCAATCAAAAACTTACCCAATCCA-3′. The PCR products were purified using illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare). Purified PCR products were used for TA cloning into pT&A^TM vector (Yeastern Biotech). Ten independent clones in each case were sequenced.

To determine whether VP1 sequences could be generated using RNAs recovered from the LFD, a one-step RT-PCR was performed [13] utilising forward primers O-1C244F (5'-GCAGCAAAACACATGTCAAACACCTT-3') or O-1C272F (5'-TBGCRGGNCTYGCCCAGTACTAC-3') and reverse primer EUR 2B-52R (5'-GACATGTCCTCCTGCATCTGGTTGAT-3') on elution wash collected from LFDs which had been stored at RT for one month. After PCR, amplification products were analysed by electrophoresis on a 1.5% agarose gel. Fragments of appropriate size (O-1244F/EUR2B-52R – 1162-1165 and O-1272F/EUR2B-52R – 1132-1135 nt) recovered from the gel were purified (Illustra GFX PCR DNA and Gel Band Purification Kit, GE Healthcare Life Sciences, UK) as per the manufacturer's instructions prior to sequencing as previously described [13] . The raw data were assembled using the Lasergene 11 suite (DNASTAR, Madison, WI) and further sequence analysis performed using BioEdit (version 7.0.1) [14] .

The amplified fragments were purified using the Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare) according to the manufacturer's protocol and sequenced directly at Genomic (Genomic Engenharia Molecular, Sao Paulo, Brazil). DNA sequencing was performed on an Applied Biosystems 3130xl DNA analyzer using the BigDye Terminator v3.1 cycle sequencing kit.

The concentration of DNA was measured using qPCR [38 (link)] and normalized to 2 ng per PCR reaction. The V4 region of the 16S rRNA gene was amplified [39 (link)] with primers containing the respective Illumina adapters and a unique 8-nt index sequence key [40 (link)]. The amplification was performed according to Kozich et al. [40 (link)], with the exception of 33 cycles instead of 35. The amount of DNA per sample was quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Thermo Fisher Scientific), pooled equimolarly, and purified using the Illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Eindhoven, the Netherlands). The quality and size of the amplicons was analyzed on the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Paired-end sequencing (200 cycles) of the DNA was conducted on the MiSeq platform (Illumina, San Diego, CA, USA) at TNO (Zeist, the Netherlands). The flow cell was loaded with 6 pmol DNA containing 50 % PhiX. The sequence data have been submitted to the Sequence Read Archive (SRA) under accession number prjna308439.

Genomic DNA was extracted using the UltraClean® Microbial DNA Isolation kit (Mo Bio, CA, USA). The integrity and quantification of genomic DNA were evaluated by fluorimetry using the Qubit 2.0 Fluorimeter® (Invitrogen). The partial sequence of the CaM gene was amplified with the primers CMD5 (CCGAGTACAAGGARGCCTTC) and CMD6 (CCGATRGAGGTCATRACGTGG) according to Hong et al. (2005 (link)). The reactions were prepared in a final volume of 50 μl with the following reagents and concentrations: 60 ng of DNA of each sample, 1 × dAmpliTaq Gold® 360 Master Mix (Life Technologies) and 0.5 pmol/μl of each primer (forward and reverse).
Successfully amplified PCR products were purified using the Illustra® GFX PCR DNA and Gel Band Purification kit (GE Healthcare Life Sciences) and sequenced on an ABI3130 automated sequencer (Applied Biosystems, Life Technologies Q7, CA, USA). The sequences were manually edited using Geneious software (version 9.1.6) (Kearse et al., 2012 (link)) and deposited into the NCBI GenBank (Table 1).