Exosap it express pcr product cleanup kit

MES13 control cells and Ca_V3.1, Ca_V3.2 SKO and DKO clones were subjected to genomic DNA extraction by using the QIAamp DNA extraction kit (Qiagen Ltd, Crawley, UK), following the manufacturer’s instructions. The genomic DNA was quantified in a Nanodrop, and 100 ng of DNA was amplified by using Dream Taq polymerase and primers spanning the gRNA sequence region in 20 µl reaction mix. The primer list is detailed in Suppl. Table S1. The PCR products were cleaned using the ExoSAP-IT Express PCR product clean-up kit (Thermo Fisher Scientific). The PCR product was preceded to Sanger sequencing by GATC Biotech, Ebersberg, Germany.

The PCR products obtained were purified using the ExoSAP-IT Express PCR Product Cleanup Kit from Thermo Fisher (Santa Clara, USA). The purified PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (Austin, USA), carried out on a 4 capillary 3130 Genetic Analyzer provided by Thermo Fisher.
To analyze the nucleotide sequences, a comparative analysis was conducted using the BLAST program (NCBI, USA), which is available at https://blast.ncbi.nlm.nih.gov/Blast.cgi. The sequences were compared to existing sequences in the database to identify similarities and relationships.
The evolutionary history of the sequences was inferred using the Neighbor-Joining method. The evolutionary distances between sequences were calculated using the Maximum Composite Likelihood method, expressed as the number of base substitutions per site. The analysis included codon positions of 1^st, 2^nd, 3^rd, and non-coding regions. Any ambiguous positions in the sequences were removed (pairwise deletion option) before conducting the analysis.
The phylogenetic analysis was performed using MEGA11 (https://www.megasoftware.net/), a widely used evolutionary analysis tool [23 (link)].

PCR products with bands at the expected sizes were enzymatically cleaned up using the ExoSAP-IT™ Express PCR Product Clean Up kit (Thermo Fisher Scientific^®, Berlin, Germany). Sanger sequencing was performed directly on both strands at the sequencing facility of the Robert Koch Institute, Berlin, Germany, using 0.5 µl Big Dye version 3.1 (Life Technologies, Applied Biosystems, Darmstadt, Germany). The resulting aHEV sequences were compared with reference aHEV strains available at GenBank. Multiple sequence alignments with reference strains were performed using the MAFFT method with default options in Geneious Prime software version 2020.0.5 (Biomatters, New Zealand). Phylogenetic trees were constructed by maximum likelihood method with 1000 standard non-parametric bootstrap replicates using IQ-TREE 1.6.12 [42 (link)]. The best fitting model was determined using the substitution model test included in IQ-TREE [43 (link)]. Trees were graphically adjusted using iTOL [44 (link)].