Dreamtaq dna polymerase mastermix

Linear DNA encoding for the fluorescent protein mVenus was prepared by Polymerase Chain Reaction (PCR) as reported previously^{3 (link)}. PCR reactions were carried out using DreamTaq DNA polymerase MasterMix (2x, ThermoFisher), Forward and Reverse Primers (Supplementary Table 1, Entry 1 and 2) at 0.25 µM concentration and 0.04 ng/µL of the HindIII-digested mVenus plasmid as template in a total reaction volume of 50 µL. The PCR was carried out according to the manufacturer’s protocol for 35 cycles with an annealing temperature of 47 °C for 30 s, an extension time of 72 °C for 1 min (15 s/kbp) and a final extension at 72 °C for 10 min. The resulting DNA was then purified using the GeneJet PCR purification columns (ThermoFisher) following the manufacturer’s protocol and eluted in 50 µL H₂O.

All consumables, solvents and reagents were purchased from Sigma/Merck unless stated otherwise. NHS–PCB–Biotin was purchased from Ambergen and stored in dry dimethylformamide at −80 °C. DreamTaq DNA polymerase master mix, TR-dextran (10 kDa) and 25 µl gene frames were purchased from Thermo Fisher Scientific. PURExpress and all other enzymes were purchased from New England Biolabs. E. coli XL10-Gold cells were purchased from Agilent. Egg PC was purchased from Avanti Polar Lipids. The 1 mm diamond drill bit was purchased from Eternal Tools. The M365L2-C5 UV lamp was purchased from Thorlabs. Standard DNA oligonucleotides were synthesized by integrated DNA technologies. Photomasks were designed using AutoCAD and purchased from JD Photodata. Amine-modified DNA oligonucleotides were synthesized by ATD-Bio. pMAT–mNG was synthesized by GeneArt. pSB1A3–bjaR–gfp was a gift from K. Haynes (Arizona State University). pET24a was a gift from B. Davis (University of Oxford). Monovalent streptavidin was a gift from M. Howarth (University of Oxford). All DNA sequences can be found in Supplementary Data.

First, 1.5 × 10⁵ HEK293H cells were seeded in each cavity of a 24-well plate one day prior to transfection with the pDESTsplice reporter constructs. Cells were transfected using Lipofectamine LTX Reagent according to the manufacturer’s instructions. After 4 h, a medium change was performed. After a total of 48 h post-transfection in culture, cells were washed with PBS, dissolved in RNA Lysis Buffer, and RNA was isolated using the associated Quick-RNA Miniprep Kit (Zymo Research, Freiburg i. Breisgau, Germany). RNA concentration was determined in a Spark Microplate Reader (Tecan, Männedorf, Switzerland) equipped with a NanoQuant Plate. Subsequently, 500 ng of the obtained RNA was transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Braunschweig, Germany). Polymerase chain reaction was performed using DreamTaq DNA Polymerase Mastermix (Thermo Fisher, Braunschweig, Germany) with the addition of the template cDNA and the desired primer pair (Table S3). The PCR result was applied to a 1.5–2% TAE agarose gel to which Sybr Safe (Thermo Fisher, Braunschweig, Germany) was added as DNA band staining. Bands were captured at 600 nm in the Odyssey XF imager (LI-COR Biosciences, Bad Homburg v. d. Höhe, Germany) and analyzed using Empiria Studio 2.2.