Hot firepol dna polymerase

Genomic DNA was extracted using Wizard Genomic DNA Extraction Kit (OMGA-Bio-Tek, Inc., Madison., WI, USA). DNA quality was tested using 1.5% agarose gel electrophoresis. Polymerase chain reaction (PCR) was used for amplifying the studied genes; using primers targeting exon II of the β-LG, intron 2 of PRL as shown in Table 1. Primers for CSN3 were designed, using primer 3 (http://frodo.wi.mit.edu/primer3/), to target part of the intron 3 and the totality of exon 4 and part of intron 4, using the available nucleotide sequence (Accession No.: 443394) on the NCBI GenBank database. PCR mix (HOT FIREPol DNA Polymerase; Solis BioDyne, Estonia) was carried out in a total volume of 20 μLcontaining10μL of nuclease-free water, 2 μL of genomic DNA (100 ng/ μL) as a template, 2 μL of each primer, and 4 μL (5U/µL) of Taq DNA polymerase (Eppendorf AG, Hamburg, Germany). Primer sequences, annealing temperature, and restriction enzymes used for genotyping are shown in Table 1. The PCR reaction was carried out in the following conditions of 95 °C for 5 min for initial denaturation followed by 33 cycles at 95 °C for 30 s of denaturation, 40 s annealing (Table 1) and extension each at 72 °C, and a final extension step at 72 °C for 7 min.

The poorly covered regions from NGS were amplified and verified by Sanger sequencing. The variants identified by multiplex genetic sequencing were also validated with Sanger sequencing. In brief, primers were designed using Primer3 software [16 (link)]. Primer sequences for validation are listed in S2 Table. Identified variants, including WRN (c.4108DelA), DICER1 (c.A3334G), and ELAC2 (c.A248G), were amplified in duplicate from genomic DNA of the six family members by using Hot FirePol DNA polymerase (Solis BioDyne, Tartu, Estonia). Sanger sequencing was performed using the Big Dye Terminator Cycle v1.1 Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA) and ABI Prism 3130 Genetic Analyzer (Applied Biosystems).

Cells were transfected with plasmid expressing Cas9 and gRNA (either hTRIB3-KO-guide1 and hTRIB3-KO-guide2 mixture, or AAVS1-KO-guide1) using the Lipofectamine 3000 transfection reagent (Thermo Scientific) and Opti-MEM I Reduced-Serum Medium (Thermo Scientific) supplemented with 3% FCS. 8 h post-transfection the medium was changed to complete growth medium. The next day, cells were seeded into either 96-well or 12-well plates and cultured for 24 h, after which the cells were used for luciferase reporter assays, for Trypan blue cell viability assays and for measuring mRNA or protein expression.
Genomic DNA was extracted from cells 24 h after transfection and the genomic region containing the CRISPR target site in the TRIB3 gene was amplified with HOT FIREPol DNA Polymerase (Solis BioDyne, Tartu, Estonia) using primers #1050 and #1051 (Table S4). The PCR products were resolved on 1.8% agarose gel, stained with ethidium bromide and visualized by UV light to evaluate the fraction of alleles with a targeted deletion (excision of the gRNA1–gRNA2 fragment).