We isolated plasmid DNA from each FACS cell population and the time points from the growth experiment as described (Jiang et al., 2013 (link)). Additionally, we sequenced the original barcoded plasmid library to evaluate the collateral effects on variants during the pre-selection library expansion stages. Purified plasmid DNA was linearized with AscI. Barcodes were amplified with 22 cycles of PCR using Phusion polymerase (NEB) and primers that add Illumina adapter sequences and a 6 bp identifier sequence used to distinguish cell populations. PCR products were purified two times over silica columns (Zymo Research) and quantified using the KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems) on a Bio-Rad CFX machine. Samples were pooled and sequenced on an Illumina NextSeq instrument in single-end 75 bp mode.
Silica columns
Manufactured by Zymo Research
Silica columns are a type of laboratory equipment used for the purification and separation of various biomolecules, such as DNA, RNA, and proteins. These columns contain a silica-based stationary phase that interacts with the target molecules based on their specific properties, allowing for their selective retention and subsequent elution. Silica columns are commonly used in applications like solid-phase extraction, affinity chromatography, and nucleic acid purification.
Automatically generated - may contain errors
3 protocols using silica columns
1
Plasmid DNA Isolation and Sequencing
2
Capillary Electrophoresis and Gel Analysis
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Residual primers were removed on silica columns (Zymo Research). cDNAs generated with HEX-labeled primers or FAM-ddUTP were analyzed by capillary electrophoresis (Genewiz). cDNAs generated with IRD-700-labeled primers were separated on 6% polyacrylamide urea gels and analyzed by infrared laser scanning (Odyssey, LI-COR).
3
Plasmid DNA Isolation and Barcode Amplification
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We isolated plasmid DNA from each bulk competition time point as described (Jiang et al., 2013 (link)). Purified plasmid was linearized with AscI. Barcodes were amplified by 19 cycles of PCR using Phusion polymerase (NEB) and primers that add Illumina adapter sequences and an 8 bp identifier sequence used to distinguish libraries and time points. The identifier sequence was located at positions 91–98 relative to the Illumina primer and the barcode was located at positions 1–18. PCR products were purified two times over silica columns (Zymo Research) and quantified using the KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems) on a Bio-Rad CFX machine. Samples were pooled and sequenced on an Illumina NextSeq instrument in single-end 100 bp mode.
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