Journal: Medical research archives

Article Title: Correspondence of Yolk Sac and Embryonic Genotypes in F0 Mouse CRISPants

doi: 10.18103/mra.v11i6.3989

Figure Lengend Snippet: Fertilized mouse oocytes were microinjected with guide RNA and Cas9 protein, and transferred into pseudo-pregnant foster dams. Conceptuses were recovered at day E10.5, visceral yolk sac was dissected away, and the embryo appearance was inspected under a stereo microscope before samples were processed for genomic DNA preparation and sequencing. Panel A displays the relative representation of each distinct allele in a pie chart for each sample, consistent with the color scheme in ; wildtype is shown in black. Pie charts are arranged in the order of presentation in the Results section of this manuscript, in pairs of yolk sac and embryo for each conceptus. Panels B-E depict the morphology of the respective embryos to the right: B: 29N-A; C: 29N-F; D: 29N-E, and E: 29N-G1.

Article Snippet: Libraries were generated using the PCR barcoding (96) genomic DNA Kit (SQK-LSK109) and sequenced on the MinION Portable Sequencer (Oxford Nanopore Technologies) .

Techniques: Microscopy, Sequencing

Journal: bioRxiv

Article Title: A Generalized Platform for Artificial Intelligence-powered Autonomous Protein Engineering

doi: 10.1101/2025.02.12.637932

Figure Lengend Snippet: The site-directed mutagenesis protocol used a 27 bp mutagenic primer (12+3+12) for generating mutations in the ORF. Each mutation site required two mutagenic primers to amplify the adjacent fragments. Another set of primers were used for PCR of vector backbone. The three PCR fragments contained overlapping regions of 27–40 bp. The template plasmids for PCRs were linearized with appropriate restriction enzyme. For ORF PCR, ∼200 pg of template DNA was used per reaction. The PCR reactions were treated with Dpn I to eliminate the original templates, followed by HiFi assembly of the backbone and two adjacent ORF fragments.

Article Snippet: For PCR templates, the plasmids were linearized with restriction enzymes and purified using a PCR cleanup kit (Zymo #D4018).

Techniques: Mutagenesis, Plasmid Preparation

Journal: bioRxiv

Article Title: A Generalized Platform for Artificial Intelligence-powered Autonomous Protein Engineering

doi: 10.1101/2025.02.12.637932

Figure Lengend Snippet: Module 1 of the workflow prepared mutagenesis PCR for 96 mutants in a 96-well PCR plate. The templates and primers for PCR were mixed using worklists on the Tecan Fluent and Echo liquid handler. PCR success was measured by adding 2.5 μL of the PCR reaction to 47.5 μL of 1x Evagreen dye (Biotium #31000) and measuring fluorescence ( λ ex = 498 nm / λ em = 535 nm) with the Tecan Infinite plate reader. 25 μL PCR product was transferred to a new PCR plate, and 1 μL of Dpn I (NEB #R0176) was added, followed by incubation at 37 °C. The Dpn I-treated PCR products were then transferred to a 384-well plate, along with the vector backbone, and a worklist guided the mixing of the correct fragments in the Echo liquid handler for HiFi assembly. After a 30-minute HiFi assembly at 50 °C, competent DH5α cells in a 96-well plate were transformed by heat shock on the Tecan Fluent using onboard heating/cooling blocks. The cells were plated on 8-well omnitray agar plates containing LB + 50 μg/mL kanamycin and incubated overnight at 37 °C in a Cytomat automated shaking incubator.

Article Snippet: For PCR templates, the plasmids were linearized with restriction enzymes and purified using a PCR cleanup kit (Zymo #D4018).

Techniques: Mutagenesis, Fluorescence, Incubation, Plasmid Preparation, Transformation Assay