Maxima hot start dna polymerase

Targeted loci were amplified from the genomic DNA by PCR using the Maxima Hot Start DNA polymerase (Thermo Fischer Scientific) according to the manufacturer’s instructions. The PCR primers (S3 Table) were designed to anneal upstream and downstream of the expected cutting site. The PCR product was purified using Exo I and FastAP (Thermo Fischer Scientific) treatment 15min 37°C, then 15min 85°C. 10μl of the purified PCR product was annealed in a reaction containing 1x NEBuffer 2 (New England Biolabs, MA, USA) and was run on a 10% polyacrylamide gel. The gel was stained with GelRed (Bitium Inc., Fremont, CA).

RNA was isolated from basophils with a purity ≥99% using the “Direct-zol RNA MiniPrep Kit” from Zymo Research and reversely transcribed into cDNA using the “RevertAid H Minus First Strand cDNA synthesis Kit” (Thermo Scientific). A minimum of 5 × 10⁶ basophils was required to obtain sufficient amount of RNA. The RNA was either stored at −80°C or subjected immediately to the reverse transcription reaction. DNA concentration and quality was controlled with the Nanodrop photometer (Peqlab). Specific TLR cDNA was amplified by the following PCR protocol: pre-denaturation step at 95°C for 15 min followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, followed by a final extension step at 72°C for 10 min before cooling to 4°C. Amplification was performed using the Maxima Hot Start DNA Polymerase (Thermo Scientific). Table 1 shows the sequences of the applied TLR-specific primers. A negative control reaction was performed without cDNA template and a positive control using specific primers for the housekeeping gene GAPDH. PCR products were analyzed on 1% agarose gels together with the “MassRuler DNA Ladder Mix” from Thermo Scientific (marker 1) and the “100 bp ladder” from Promega (marker 2).

Targeted loci were amplified from genomic zebrafish DNA by PCR using Maxima Hot Start DNA polymerase (Thermo Scientific) according to manufacturer’s instructions. PCR primers (Table 3) were designed to anneal upstream and downstream of the expected cutting site. A previously described T7 endonuclease assay protocol with minor changes was used [35 ]: 1.5μg of PCR product was annealed in a 20μl reaction of 1x NEBuffer 2 (New England Biolabs, MA, USA) and the annealed sample was incubated with 0.5μl (6 units) of T7 endonuclease I (New England Biolabs) for 30 minutes at 37°C. Obtained products were separated with a 2.5% agarose TAE gel and the band sizes were compared to control samples.