Libraries were prepared for deep sequencing as described in Medina-Cucurella and Whitehead (Medina-Cucurella and Whitehead 2018 (link)), using a Zymo Yeast Plasmid Miniprep II kit (Zymo Research) and a Monarch PCR & DNA Cleanup kit (NEB) with the following changes. Inner primers RBD-F1_tile1_F_Illumina and RBD-F1_tile1_R_Illumnia were used to amplify tile 1 with an annealing temperature of 70°C; inner primers RBD-F1_tile2_F_Illumina and RBD-F1_tile2_R_Illumnia were used to amplify tile 2 with an annealing temperature of 63°C. 5 μL of PCR product from the inner primer amplification was cleaned used 0.5 μL Exonuclease I (NEB) and 1 μL rSAP (NEB), incubating for 15 min at 37°C then 15 min at 80°C. 2 μL of purified DNA was carried forward to the 2nd PCR reaction. Samples were purified using Agencourt Ampure XP beads (Beckman Coulter), quantified using PicoGreen (ThermoFisher), pooled, and sequenced on an Illumina MiSeq using 2 × 250 bp paired-end reads at the BioFrontiers Sequencing Core (University of Colorado, Boulder, CO). Library statistics are listed in Table S3 .
Zymo yeast plasmid miniprep 2 kit
Manufactured by Zymo Research
Sourced in United States
The Zymo Yeast Plasmid Miniprep II kit is a laboratory product designed for the rapid and efficient isolation of plasmid DNA from yeast cells. It is a tool used in molecular biology and genetic research applications.
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Lab products found in correlation
Monarch pcr dna cleanup kit, by New England Biolabs (1 mentions)
Picogreen, by Thermo Fisher Scientific (1 mentions)
Exonuclease 1, by New England Biolabs (1 mentions)
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
Snap surface alexa fluor 488, by New England Biolabs (1 mentions)
In fusion cloning kit, by Takara Bio (1 mentions)
Expi293 cell, by Thermo Fisher Scientific (1 mentions)
Frozen ez yeast transformation 2 kit, by Zymo Research (1 mentions)
3 protocols using zymo yeast plasmid miniprep 2 kit
1
Amplification and Sequencing of RBD Tiles
2
Yeast Plasmid Extraction and Quantification
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Following sorting, yeast were pelleted by spinning at 3000 × g for 10 min at 4°C. Supernatant was carefully removed by pipette, and the resulting pellet was resuspended in 4 mL SDCAA and transferred to a glass culture tube. A small amount of this resuspension (targeting 200–500 cells, based on sorting counts) was plated on SDCAA-agar and YPD-agar to quantify recovery efficiency and plasmid loss. Cultures were then rocked at 30°C until reaching OD600 = 0.8–2.
After reaching the target OD600, 1.5 mL yeast culture was pelleted and frozen at –80°C for at least an hour. Plasmid was then extracted using the Zymo Yeast Plasmid Miniprep II kit (Zymo Research #D2004) following the manufacturer’s instructions, except for the following changes: 5 µL zymolyase was used per sample, zymolyase incubations were 2–3 hr, precipitate following neutralization was removed by centrifugation at 21,000 × g for 10 min, columns were washed using 650 µL wash buffer and dried by spinning at 16,000 × g for 3 min, and plasmid was eluted in 15 µL elution buffer.
After reaching the target OD600, 1.5 mL yeast culture was pelleted and frozen at –80°C for at least an hour. Plasmid was then extracted using the Zymo Yeast Plasmid Miniprep II kit (Zymo Research #D2004) following the manufacturer’s instructions, except for the following changes: 5 µL zymolyase was used per sample, zymolyase incubations were 2–3 hr, precipitate following neutralization was removed by centrifugation at 21,000 × g for 10 min, columns were washed using 650 µL wash buffer and dried by spinning at 16,000 × g for 3 min, and plasmid was eluted in 15 µL elution buffer.
3
Yeast Display of Human FcRn Receptor
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