Wizard sv gel and pcr clean up system kit

The plasmids were purified from transformed E. coli DH5α cells using the Wizard® Plus SV Minipreps DNA Purification System Kit (Promega, USA), and then cut with restriction enzymes from Invitrogen (USA) or New England Biolabs (USA). The DNA fragments were purified with the Wizard® SV Gel and PCR Clean-Up System Kit (Promega, USA) from agarose gels between 1 to 1.2% (w/v) in TBE buffer. Genomic DNA obtained from CHO-K1 (ATCC® CCL-61™) cells was purified using the PureLink™ Genomic DNA Mini Kit (Invitrogen, USA). The PCR reactions for DNA fragments analysis were carried out with the GoTaq® Green Master Mix Kit (Promega, USA). The PCR reactions to generate cloning fragments were performed with KAPA HiFi HotStart high-fidelity DNA polymerase (Kapa Biosystems, USA).

Analysis of GBA/GBAP1 splicing patterns and susceptibility to NMD was undertaken in HepG2 and HEK293 cell lines. Cells were plated at a density of 4 * 10⁵ per 6-well dish and, after 72 hours, treated for 8 hours with cycloheximide (100 µg/mL; dissolved in dimethyl sulfoxide) or with the vehicle alone. After the treatment, cells were washed with PBS and total RNA extracted.
For the analysis of the splicing pattern, a set of gene-specific or pseudogene-specific RT-PCR assays (Supplementary Table 3) was designed to catch the vast majority of possible alternative splicing events. RT-PCRs were performed as described above. The main amplified products, recovered from the agarose gel using the Wizard SV Gel and PCR Clean-Up System kit (Promega), were directly sequenced to confirm their identity.
Variations in the expression levels of GBA/GBAP1 upon treatment were quantified by real-time RT-PCR assays using as reference an NMD-resistant transcript (i.e., Connexin 43 or Connexin 32 mRNAs, whose coding sequences are all contained in a single exon, for HEK293 and HepG2, respectively). The NMD-sensitive and insensitive PRKCA transcripts were used as controls^{32 (link)}.

Amplified target DNA bands were either purified directly from the PCR reaction or from the gel using Wizard® SV Gel and PCR Clean-Up System kit (Promega Madison, WI, USA). Sequencing was outsourced and performed using ABI-PRISM 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). Both forward and reverse strands were sequenced and the raw chromatogram file was edited for bad calls using DNAbaser v.3.0.The sequences were compared with available sequences using Basic Local Alignment Search Tool and the GenBank database to confirm the identity of the virus isolate. The sequences were aligned using Muscle [24 (link)] in Molecular Evolutionary Genetics Analysis (MEGA) software version 76 was used for phylogenetic analysis using the Maximum likelihood statistical method tested with 1000 bootstrap replicates based on the Tamura-Nei model [25 (link)]. The phylogenetic tree was inferred in MEGA version 7. A total of 15 (5 for each serotype DENV1-3) samples were sequenced.