Generuler low range dna ladder

Following the PCR, samples were tested for amplification success by gel electrophoresis using a 2% agarose gel (Sigma-Aldrich; A6877) in TAE buffer (AppliChem GmbH; A1691) at 4 °C. With a volume of 100 ml, the gel contained 4 µL of the dye peqGreen (VWR International GmbH; 37–500). For the loading of the agarose gel, 5 µL of the unpurified PCR product was mixed with 1 µL of 6× DNA Loading Dye (Thermo Fisher Scientific Inc.; R0611). The molecular weight marker used was composed of 1 µL Gene Ruler Low Range DNA Ladder (Thermo Fisher Scientific Inc.; SM1191), 1 µL 6 × TriTrack DNA Loading Dye (Thermo Fisher Scientific Inc.; R1161) and 4 µL nuclease-free water. Electrophoresis ran at a constant voltage of 100 V for 90–120 min (depending on the volume of the gel). The gel electrophoresis results were documented using the Quantum CX5 gel documentation system from Vilber Lourmat Deutschland GmbH (Eberhardzell, Germany) and the related BioVision software.

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.

Phi29 polymerase, phi29 buffer, dNTP mix, bovine serum albumin (BSA), GeneRuler low range DNA ladder, SYBR Gold nucleic acid gel stain, Tris-HCl buffer (1 M, pH 8.0) and Tris-acetate-EDTA buffer (TAE, 50 ×) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). CircLigase II ssDNA ligase, together with other ligation reagents (buffer, MnCl₂, betaine), was purchased from Biosearch Technologies (Novato, CA, USA). Nb.BtsI nickase, thermolabile exonuclease I, exonuclease III and loading buffer were purchased from New England BioLabs (Ipswich, MA, USA). Recombinant Acidaminococcus sp. BV3L6 Cas12a nuclease was purchased from Integrated DNA Technologies (Coralville, IA, USA). Fetal bovine serum (FBS), salmon sperm DNA and agarose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Streptavidin-coated cross-linked starch iron oxide composite particles (100 nm size MNP) were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany). DNA and RNA sequences were synthesized by Integrated DNA Technologies and diluted in 50 mM Tris-HCl (pH 8.0). Sequences of targets (target dengue sequence and target B for amplifying detection loop and reference loop, respectively), linear templates (linear detection template and linear reference template), crRNA and detection probes (DP-DL-I, DP-DL-II, DP-RL-I and DP-RL-II) are listed in Supplementary Table S1.

For gel electrophoretic analysis, 3 µl DNA Gel Loading Dye (6 x) (Thermo Fisher Scientific; R0611) were added to the purified (and digested) samples except the RsaI approach. For band visualization, a 2% agarose gel was prepared in 1 × TAE buffer (50 × TAE buffer; PanReac AppliChem; A1691) containing 4 µl peqGreen (VWR; 732-3196) per 100 ml gel. 12–15 µl of the purified and prepared samples were used for electrophoresis at 120 V for 1 h before detection via BioDoc Analyzer (Biometra) using a Canon EOS 1100D. GeneRuler Low Range DNA Ladder (Thermo Fisher Scientific; SM1191) and peqGOLD DNA ladder (VWR; 25-2040) were used for calculation of fragment length.
For the detection of incorporated Cy5-dUTP the DNA gel was scanned with a Tecan LS reloaded scanner using a 3D-printed gel tray.