Zymo dna clean and concentrator 5

All mRNA generating plasmids were digested with PvuII-HF (NEB R[Δ4]151 L) and ApaLI-HF (NEB R0507L) at 37 °C for 3 h. After digestion, templates were run on a 1% agarose gel to confirm cutting and the reactions were purified with Zymo DNA Clean and Concentrator-5 (Zymo Research D4013). 1ug of linearized template was used in a 100 uL T7 RNA Polymerase (purified in house) reaction that was incubated at 37 °C for 3 h. After T7 reactions were complete, 15uL TurboDNAse (ThermoFisher Scientific AM2238) was added, and reactions were incubated at 37 °C for 15 min. The RNA was then purified using a Zymo RNA Clean and Concentrator-25 Kit (Zymo Research R1017). Eluted RNA was measured using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific Q32852) then capped following the protocol for the Vaccinia Capping System (NEB M2080S) and purified one last time using Zymo RNA Clean and Concentrator-5 (Zymo Research R1013). Capped RNA concentrations were measured using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific Q32852). Final RNA was diluted to 0.25pmoles/ul for use in the in vitro translation assays.
For the comparing capped versus uncapped mRNA, uncapped RNA was incubated for 5 min at 65 °C to match the treatment of capped RNAs. The same RNA that was used in the vaccinia capping reaction was directly compared to the post-cap RNA.

For the cfDNA elutions (see previous section) that were split, the elution half intended for ddPCR was first cleaned using Zymo DNA Clean and Concentrator-5 (Zymo Research, Irvine, CA, USA) using standard procedures and eluted twice in 10 μL of Zymo DNA Elution buffer. The elutions were then combined and DNA concentration was measured using the Invitrogen Qubit 3.0 Fluorometer and stored at − 80 °C.
To calculate the genomic copies of mutated KRAS present in cfDNA extracted from plasma samples, we used the BioRad ddPCR KRAS G12/G13 Screening Kit (which targets 7 different KRAS mutations G12A, G12C, G12D, G12R, G12S, G12V, G13D) (BioRad product number: 1863506) (BioRad, Hercules, CA, USA), using standard instructions with a C1000 Touch Thermo Cycler and the QX200 Droplet Generator and Reader System and an annealing temperature of 60 °C. 5 μL of a given sample elution was used as input for each reaction and at least two reactions for each sample were performed until the entire sample was used. Droplets were read on a QX200 Droplet Reader and data was analyzed using QuantaSoft software.

All chemicals were purchased from Sigma unless otherwise stated. All restriction enzymes, DNA ligases, and DNA polymerases used for cloning and PCR were purchased from New England Biolabs (NEB) unless otherwise stated. Plasmid minipreps, PCR purifications, and gel extractions were done using the Zymo Zyppy Plasmid Miniprep Kit, and Zymo DNA Clean and Concentrator 5. Genomic DNA from Y. lipolytica was extracted using the E.Z.N.A. Yeast DNA kit (Omega Biotek). All oligonucleotides and gBlocks^® were purchased from IDTDNA. Xylose assays were performed using the D-Xylose Assay Kit (Megazyme). Glucose assays were done using glucose oxidase and horseradish peroxidase enzymes purchased from Sigma.