Chromosomally deleted alleles were detected by genomic PCR using the primers listed in Supplementary Table S4 , followed by agarose gel electrophoresis and ethidium bromide staining. The deleted PCR products were then cloned into TA cloning vector using a TArget Clone -Plus- Kit (Toyobo). Sequencing was performed using an ABI 3131xl Genetic analyzer (Life Technologies) with a BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies).
Target clone plus kit
Manufactured by Toyobo
The TArget Clone -Plus- Kit is a laboratory equipment product designed for DNA cloning. It provides the core functionality of DNA amplification and purification, without further interpretation or extrapolation on its intended use.
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2 protocols using target clone plus kit
1
Detecting Chromosomal Deletions via PCR
2
Targeted DNA Sequence Validation
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After puromycin selection, cells were collected and genomic DNA was extracted using a DNeasy Blood and Tissue kit (Qiagen). Genomic PCR was performed using KOD FX Neo (Toyobo) using primers 5′-acaccaagacagacatctctgtcccttg-3′ and 5′-atccgtatccaatgtggggaac-3′ for the 5′ junction, and 5′-gcccttaattgtgagcggataac-3′ and 5′-tcagcaggtcaaggggaggaatg-3′ for the 3′ junction. The PCR products were cloned and transformed into bacteria using a TArget Clone Plus Kit (Toyobo). Subsequently, colony PCR products were used as templates for DNA sequencing.
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