Generuler ultra low range dna ladder

A qualitative assay for PLD3 nuclease activity was analyzed by denaturing polyacrylamide gel electrophoresis, as previously described by Gavin et al., 2018. Briefly, 50 μg of protein lysate was incubated with 5 μM ssDNA (5′-ACCATGACGTTCCTGATGCTAAGTATGCACTTCATCGTCAAGCA ATGCTATG∗C∗A∗T-3′, Biomers) modified with internucleotide phosphorothioate (∗) in MES buffer (50 mM MES pH 4.5, 100 mM NaCl) for the indicated time at 37 ºC. The enzymatic reaction was deactivated by incubation at 96 ºC for 5 min, and samples were mixed with 6X DNA loading dye (Thermo Fisher Scientific). DNA was separated in a vertical chamber using a 20% denaturing polyacrylamide gel in 1X Tris-Borate-EDTA (TBE) buffer (1 M Tris, 1 M Boric acid, 0.02 M EDTA) applying a current of 60 V for 6 h. The GeneRuler Ultra Low Range DNA Ladder (Thermo Fisher Scientific) was used to monitor the progress of the electrophoresis. DNA gel was incubated at room temperature for 5 min in 1X SYBR Gold Nucleic Acid Gel Stain (Invitrogen) prepared in TBE buffer. The acquisition was performed with the Odyssey FC Imaging system at 600 nm and visualized with the Image Studio Software (LI-COR).

Aliquots of the ligation reaction were analyzed on 10% TBE-urea polyacrylamid gels (Thermo Fisher Scientific, EC6875BOX) to assess ligation efficiency. 2 µl ligation reaction or low range ssRNA ladder (New England Biolabs, N0364S) were heated for 5 min to 70 °C in 10 µl Novex TBE-urea sample buffer (Thermo Fisher Scientific, LC6876) and rapidly cooled before loading. Additionally, 1 µl GeneRuler Ultra Low Range DNA Ladder (Thermo Fisher Scientific, SM1213) was used for size estimation. Gels were stained 15 min at room temperature with SYBR Green II RNA gel stain (Thermo Fisher Scientific, S7564) and images acquired on a ChemiDoc Imaging System (Bio-Rad).

Native PAGE of r(G₂C₄)₄, r(G₄C₂)₄ and equimolar mixture of r(G₂C₄)₄ and r(G₄C₂)₄ was performed on 20% polyacrylamide gel (5 °C at 100 V) in 1×TBE (pH 6.0) buffer. Samples for native PAGE were prepared by mixing 5 or 10 μL of stock or diluted RNA oligonucleotide solution, 5 μL of loading buffer 15% Ficoll and diluted to 20 μL with autoclaved H₂O. The approximate band size was determined by using the GeneRuler Ultra Low range DNA Ladder (Thermo Scientific, Waltham, MA, USA). The samples were treated at room temperature overnight before loading, except for the equimolar mixture sample, which was heated to 90 °C and cooled slowly at room temperature prior to loading. Following the overnight electrophoresis, the gel was stained with Stains-All gel stain solution (Sigma Aldrich).

PCR analysis and polyacrylamide gel electrophoresis was performed for validating antisense transcripts and splicing events. SuperScript III Reverse Transcriptase (Life Technologies) enzyme, 70 ng of total RNA and gene specific primers were used for the cDNA synthesis, according to the manufacturer’s instructions. A noRT control was used for testing the potential DNA contamination. The cDNA samples were amplified using the Applied Biosystem’s Veriti Thermal Cycler with KAPA HiFi PCR Kit (KAPA Biosystems) according to the manufacturer’s recommendations. The running conditions were as follows: 3 min at 95 °C for initial denaturation, followed by 35 cycles at 98 °C for 20 s (denaturation), at 63 °C for 20 s (annealing), and at 72 °C for 2 min (extension). Final elongation was set at 72 °C for 5 min. Primers used in this study are outlined in Supplementary Table S1. For amplicon separation and visualization, a 12% polyacrylamide gel was prepared. Lanes were loaded with either GeneRuler Ultra Low Range DNA Ladder (Thermo Fisher Scientific), or the samples. Staining was performed with GelRed (Biotium).

A temperature controlled vertical electrophoretic apparatus and TBE buffer were used for native PAGE gel (15%) electrophoresis. The temperature of cooler was 5 °C for both gels. G-wire samples were deposited on gel at 0.25 mM concentration per strand whereas of Q1k, Q2k, and Q2t G-quadruplexes were deposited on gel at both 0.10 and 0.25 mM concentration per strand (1 and 2.5 nmol). Prior to loading, samples were diluted with blank solution containing the same concentration of KCl as used in individual sample. PAGE were run in the presence of 0 and 25 mM KCl in the gel as well as in running buffer for G-quadruplex and G-wire samples, respectively. Prior to loading Ficoll was added to the samples. We used Thermo Scientific GeneRuler Ultra Low Range DNA Ladder (from 300 to 10 bp) as standard for size. Electrophoresis with G-quadruplex and G-wire samples were run at 150 V for 2.5 h and 3 h, respectively. DNA was visualized by Stains-All (Sigma-Aldrich) staining.

Size-exclusion chromatography (SEC) was conducted as described [34 (link),35 (link)] using an Agilent 1200 HPLC system with an autosampler and diode array detector (Agilent; Santa Clara, CA, USA). The HPLC column was a TSKgel G2000SWXL (Tosoh Bioscience; King of Prussia, PA, USA), 30 cm length, 0.78 cm diameter, 5 µm particle diameter, 12.5 nm pore diameter; the column was conventionally recommended by the manufacturer for the separation of proteins with MW (molecular weight) in the range 5–150 kDa. The developed conditions for the separation of oligonucleotides were the following: temperature 25 °C, mobile phase water/acetonitrile 9:1 v/v with potassium phosphate buffer (60 mM KH₂PO₄ and 140 mM K₂HPO₄, pH 6.9), and flow rate 0.5 mL/min. Absorption at 260 nm was registered with 10 nm bandwidth.
The column was calibrated with a set of duplexes (GeneRuler Ultra Low Range DNA Ladder from Thermo Fisher Scientific (Carlsbad, CA, USA). The linear range for log (MW) from Vr/V0 was achieved for duplexes with 10–100 base pairs (Vr—retention volume, V0—dead volume of the column, 5.64 mL). The following equation was acquired from the calibration experiment and used for further calculations of apparent molecular weights of GQ and complexes: log(MW) = 6.51 − 1.67 · Vr/V0.