Firepol dna polymerase

A duplex PCR protocol by [29 (link)] was used for the preparation of the reaction mixture, which was modified as follows: 2.5 µL buffer, 2 µL MgCl₂, 0.5 µL dNTPs, 0.3 µL DNA polymerase (FIREPol DNA Polymerase produced by Solis BioDyne, Tarfu, Estonia), and was used for a single sample. Then, we proceeded as instructed in their protocol. In order to diagnose species of Nosema spp., two 30 picomole primers were added to the reaction mixture, 0.6 µL to each (Nosema apis for the amplification of 321 bp) APIS FOR-5′-GGGGCCATGTCTTTGACGTACTATGTA-3′ and 321 APIS REV 5′-GGGGGGCGTTTAAAATGTGAAACAACTATG-3′ and (Nosema ceranae for the amplification of 218 bp) MITOC FOR-5′CGGCGACGATGTGATATGAAAATATTAA-3′ and 218 MITOC REV 5′-CCCGGTCATTCTCAAACAAAAAAACCG-3. 2.5 µL of DNA template was added to the mixture, and PCR water was filled to final volume of 25 µL.
PCR was performed using a VWR RISTRETTO Personal Thermocycler according to the protocol of Ostroverkhová et al., 2020. ELFO was performed using 1.5% agarose gel after PCR reaction had been completed. After assessing the results using a UV Transilluminator, the DNA concentration was measured using the NanoDrop, and the PCR products were sent for sequencing. The obtained sequences were compared with the sequences stored in the gene bank using BLAST program. https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 20 November 2022).

Plasmid DNA and genomic DNA were isolated using the peqGOLD Plasmid Miniprep Kit I and the peqGOLD Bacterial DNA kit (Peqlab Biotechnologie GmbH, Erlangen, Germany), respectively. To improve lysis of M. caseolyticus, cells were first incubated in Solution I of the kit supplemented with 50 mg/l of lysostaphin (Sigma-Aldrich, St Louis, MO, USA) and 2 g/l of lysozyme (Roche Diagnostics, Rotkreuz, Switzerland) for 20 min at 37 °C. For analytical PCR reactions, FIREPol^® DNA polymerase (Solis BioDyne, Tartu, Estonia) and GoTaq^® Long PCR Master Mix (Promega, Madison, WI, USA) were used for short (<2.5 kb) and long amplicons (up to 20 kb), respectively. Insert amplifications for plasmid construction were performed using High-Fidelity DNA polymerases (Pfu DNA polymerase [Promega] or the Phusion Hot Start II High-Fidelity DNA polymerase [Thermo Fisher Scientific, Waltham, MA, USA]) according to the manufacturer’s instructions. All relevant primers used in this study are listed in Supplementary Table S1. The presence of mecD was confirmed by PCR using primers mecD-F (5′-TCCTTTAGCGATAGATGGTGAA) and mecD-R (5′-CTCCCATCTTTTCTCCATCCT).

All study subjects (n = 2324) were typed for complete AZFa (loss of markers sY84 and sY86), AZFb (sY127 and sY134), AZFc (sY254 and sY255), and partial AZFc deletions gr/gr (sY1291), b2/b3 (sY1191), and b1/b3 (sY1161, sY1191, and sY1291) following established PCR primers (Krausz et al., 2014 (link); Lin et al., 2006 (link); Supplementary file 16). The multiplex PCR contained the final concentrations of 1× PCR buffer B1 (Solis Biodyne, Estonia), 2.5 mM MgCl₂, 2.5 mM dNTP, 2 µM PCR primers for STS markers sY1291 and sY1201, 3 µM PCR primers for STS markers sY1191, sY1206, and sY1161 (Supplementary file 16), 1U FIREPol DNA polymerase (Solis Biodyne), and 10 ng of template genomic DNA per reaction. The following PCR conditions were used: for 5 min at 95°C, followed by 32 cycles of 30 s at 95°C, 30 s at 63°C and 1 min at 72°C, final extension of 10 min at 72°C and a 4°C hold. The presence/absence of PCR products in a reaction were checked on 2% agarose gel. Lack of amplification of STS marker sY1291 (but presence of all others) was used to determine the gr/gr deletion and lack of sY1191 the b2/b3 deletion.

For transfection of constructs, Percoll (GE Healthcare, Chicago, IL, USA)-enriched synchronized mature schizonts of 3D7 parasites were electroporated with 50 µg of plasmid DNA using a Lonza Nucleofector II device (56 (link)). Transfectants were selected in medium supplemented with 3 nM WR99210 (Jacobus Pharmaceuticals, Princeton, NJ, USA), 0.9 µM DSM1 (BEI Resources, NIAID, NIH), or 2 µg/mL blasticidin S (Thermo Fisher Scientific, Waltham, MA, USA). For generation of stable integrant cell lines, parasites containing the episomal plasmids selected with WR99210 were grown with 400 µg/mL Neomycin/G418 (Sigma, St. Louis, MO, USA) to select for transgenic parasites carrying the desired genomic modification as described previously (19 (link)). Each WR-resistant parasite culture was routinely placed under neomycin selection in three independent experiments using three culture dishes each time and was followed up for 60 days to monitor the appearance of viable transgenic parasites (expected to represent parasites in which the targeted gene was disrupted). Successful integration was confirmed by diagnostic PCR using FIREpol DNA polymerase (Solis BioDyne, Tartu, Estonia). For primer sequences, see Supplemental file 1. For generation of PF3D7_1252600-cKO parasites, transgenic parasites were transfected with pSkipFlox (19 (link)) to episomally express the DiCre recombinase.

Brain microvessels were isolated from adult rat brains as published earlier (Veszelka et al., 2007 (link)). For receptor expression analysis total RNA was isolated from isolated brain microvessels and brain endothelial cells by TRI Reagent (Molecular Research Center, Cincinnati, OH, USA). One microgram of total RNA was transcribed to cDNA by Maxima First Strand cDNA Synthesis Kit (ThermoFisher, Waltham, MA, USA). Specific oligonucleotide primers were designed for the Mc1r gene (XM_006222790). Primer sets were MC1R_fwd 5′-TGCACCTCTTGCTCATCGTT-3′ and MC1R_rvs 5′-ACCTCCTTGAGTGTCATGCG-3′. The predicted length of PCR product was 160 bps. Primers for β-actin were used as internal controls (NM_031144). Primer sets were ACT_fwd 5′-TACTCTGTGTGGATTGGTGGC-3′ and ACT_rvs 5′-GGTGTAAAACGCAG CTCAGTAA-3′. The predicted length of PCR product was 150 bps. PCR was performed with FIREPol DNA polymerase (Solis BioDyne, Tartu, Estonia) in T100 thermal cycler (BioRad, Hercules, CA, USA). After initial heat inactivation (95 °C for 3 min) the following cycling conditions were applied: melting 94 °C for 15 s, annealing 50 °C for 15 s, polimerization 72 °C for 20 s (35 cycles). After a final 5 min extension at 72 °C PCR products were analyzed on 3% MetaPhor agarose gel (Cambrex BioScience, Rockland, ME, USA) and fragments were verified by capillary DNA sequencing.