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Detection Process

1.Electroporate the library into Endura electrocompetent cells (Lucigen 60242).
2.Transfer cells to shake flasks, revive at 250 rpm, 37°C for 1 hour.
3.Plate onto 20 x 15-cm LB antibiotic-resistant plates, incubate overnight at 30°C.
4.Scrape colonies and perform plasmid maxiprep using ENZA endo-free plasmid maxi kit.
5.Follow the Addgene library construction protocol to perform PCR.
6.Mix and purify the PCR products.
7.Run the samples on a 2% agarose gel, purify and recover the 260-270 bp bands from the gel.
8.Quantify the extracted sgRNA plasmid library using Qubit dsDNA HS assay kit.
9.Sequence on NovaSeq 6000 with PE150 sequencing strategy. Use MAGeCK to analyze sequencing data and determine the quality of the sgRNA plasmid library.

Test Results

More than 70% of the sequencing reads aligned to the target library, with a Gini Index <0.1 and a coverage rate of 99.9%, indicating the high quality of the prepared sgRNA plasmid library.