Longamp taq reaction buffer

Around 500 ng of (treated) RNA (messenger or total RNA) were incubated at 75°C for 5 min with 2.5 μM of targeted RT primers (Table 2). Reverse transcription was performed, using 10 U ProtoScript II (New England Biolabs) in addition with 0.5 mM dNTP mix, 10 mM DTT, 0.4 U RNase inhibitor, 1x ProtoScript II Buffer (final concentrations) and MilliQ water for 2 h at 50°C. Then, polymerase chain reaction (PCR) was performed by mixing 5 U of LongAmp Taq DNA Polymerase (New England Biolabs) with 0.4 μM of targeted P5 primer (Table 2) and 0.4 μM P7 primer from the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) in addition to 300 mM dNTP mix, 1x LongAmp Taq Reaction Buffer (final concentrations) and MilliQ water. PCR was performed for 25 cycles at a primer annealing temperature of 50°C. The amplicon obtained was analysed via 10% denaturing polyacrylamide gel electrophoresis for 40 min at 10 W. The corresponding gel area (amplicon size), determined by GeneRuler Low Range DNA Ladder (Thermo Scientific), was cut out and eluted. The gel elution was performed overnight at 25°C in 0.5 M ammonium acetate solution, followed by Nanosep filtering (0.45 μm, VWR) and subsequent ethanol precipitation.

To assess the loss of the target gene in the knockout cell lines, genomic DNA was isolated from parasites collected after 1 passage post-transfection with the DNeasy Blood & Tissue Kit (Qiagen). One hundred nanograms of genomic DNA was mixed with 0.3 mM dNTPs, 0.5 μM forward primer and reverse primer, 3% (v/v) DMSO, 2.5 units LongAmp Taq DNA polymerase (NEB), and 1x LongAmp Taq Reaction Buffer supplemented with Mg²⁺ (2 mM final, NEB). The PCR conditions were 5 min at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at 60°C, 2 min 30 s at 65°C, and a final elongation step of 10 min at 72°C. Three microliters of reaction was then run on a 1% agarose gel to assess for the presence of the expected product. Primer sequences are detailed in Supplementary Materials in Table S2.

For each sample, two reactions were set up: 1) forward primer PMS2_RNA_F and reverse primer RNA_Unv_R amplified 1.5 kb of PMS2 from cDNA and 2) forward primer PMS2CL_F and reverse primer RNA_Unv_R amplified 1.5 kb of PMS2CL from cDNA (primer sequences in Additional file 2: Table S3). PCR reactions contained 1× LongAmp Taq Reaction Buffer (NEB), 0.3 mM dNTPs, 1 μM of each forward and reverse primer, 20–70 ng cDNA, 0.1 U/μL LongAmp Taq DNA polymerase (NEB), and water up to 25 μL. Thermocycling was as follows: 94 °C for 5 min, 30 cycles of 94 °C for 30 s, annealing at 52 °C for PMS2 and 55 °C for PMS2CL, 65 °C for 2 min, followed by a final extension at 65 °C for 10 min and then a 4 °C hold. PCR products were cleaned with 1.2× SPRI beads. Amplicons were visualized with a 2% agarose gel or with the DNA 7500 kit (Agilent).

Genomic DNA was extracted from 3 million transfected cells using the DNeasy Blood and Tissue kit and quantified by Quant-iT^™ PicoGreen^™ dsDNA Assay kit. To investigate the integrity of the integrated vector LTatCL[M], near full-length amplification of the vector was performed, using genomic 300 ng DNA, 1x Long Amp Taq Reaction Buffer (NEB), 0.4 mM dNTPs, 0.5 μM of each forward and reverse primer (Table S1) and 2.5 U Long Amp Taq Polymerase (NEB) in a total volume of 25 μL. The PCR cycling conditions were as follows: 94 °C for 30 min; 30 cycles of (94 °C for 20 s, 58–61 °C for 30 s, 72 °C for 5 min); 72 °C for 10 min; 4 °C hold. A total of 1 ng purified amplicons were processed with the Nextera XT DNA Library Preparation Kit (Illumina) and subsequently sequenced using the MiSeq reagent Kit v2 as described above.

The open reading frame (ORF) of HzAPN1 was PCR-amplified from the plasmid HzAPN1-pGEMT (Zhang & Li, unpublished manuscript) with the gene-specific primers APN-5′ASCI (5′-GGCGCGCCGACCAGCGTGGAGTCACAA-3′) and APN-3′NOTI (5′-GCGGCCGCTTAAGCCATATTAACAACGAGAGTCA-3′). PCR reaction (20 μL) contained 1 μL of HzAPN1-pGEMT (Zhang & Li, unpublished manuscript), 1 μL of LongAmp® Taq DNA polymerase (New England Biolabs, Ipswich, MA), 1.6 μL of dNTP (5 mM), 2 μL of primer mix (25 mM each), 4 μL of 5× longAmp Taq reaction buffer and 10.4 μL ddH₂O. The PCR reaction was initiated at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s and 65 °C for 1 min, and a final extension of 5 min at 65 °C. The PCR products were TA-cloned into pGEM®-T vector (Promega, Madison, WI), released by double digestion with ASCI and NOTI (New England Biolabs, Ipswich, MA), and subcloned into the expression vector pAc/V5-His A (Invitrogen) via the ASCI and NOTI enzyme sites.

For each virus, the whole genome of segment A was amplified by PCR using the forward primer (Seg A For, 5′-CGAGCTCGGTACCTAATACGACTCACTATAGGATACGATCGGTCTGACCCCGGGGG-3′) and the reverse primer (Seg A Rev, 5′-GGCTAAGATATCAGGGGACCCGCGAACGGGTCCAATTTGGATGTTGTATGGC-3′), yielding a product size of 3.5 kb. Similarly, the 2.8 kb segment B was PCR-amplified using the forward primer (Seg B For, 5′-GAGCTCGAATTCTAATACGACTCACTATAGGATACGATGGGTCTGACCCTCTGGG-3′) and the reverse primer (Seg B Rev, 5′-GGCTAACCGCGGGGGGGCCCCCGCAGGCGAA-3′). Briefly, a 25 μL PCR mixture was set up, containing 2 μL of cDNA template, 400-nM forward and reverse primer, 300 μM dNTPs, 5 μL of 5X LongAmp Taq Reaction Buffer, and 1 μL (2.5U) of LongAmp Taq DNA Polymerase (NEB, Ipswich, MA, USA). PCR was conducted at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, and 65 °C for 4 min 30 s, and a final extension at 65 °C for 10 min. A 1% agarose gel electrophoresis was used to visualize the 3.5 kb and 2.8 kb amplicons. Amplicons matching expected sizes were then purified from the electrophoresis gel utilizing the PureLink Quick Gel Extraction Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The purified products were ligated into the pGEMT easy vector (Promega, Madison, WI, USA) using the standard procedure for sequencing.

The DNA is anchored to the surface of a coverslip at one end and attached to a polystyrene bead at the other end (Fig. 1c) or attached to two different polystyrene beads (Fig. 5). To enable the specific binding of DNA to those surfaces, the substrate was produced with one biotin- and one digoxigenin-containing terminal. This DNA substrate was prepared by PCR amplification of λ-DNA using primers modified with 5′ biotin (5′-bio-ACTTCGCCTTCTTCCCATTT-3′) and 5′ digoxigenin (5′-dig-ATCTCGCTTTCCACTCCAGA-3′) (Eurofins MWG/Operon). The PCR reaction was performed in a 1x LongAmp Taq reaction buffer, with a final volume of 50 μl, 300 µM of each dNTP, 0.4 µM of each primer, 2 units LongAmp Taq DNA polymerase (New England Biolabs) and 0.1 ng/µl of λ-DNA template. The PCR protocol included an initial denaturation step at 94 °C for 3 min, followed by 35 cycles of denaturation (94 °C for 15 s), annealing (60 °C for 60 s) and extension (65 °C for 16 min), followed in the end by a final extension step at 65 °C for 10 min.