Abstract
Temperature-sensitive (ts) mutants provide powerful tools for investigation of cellular functions of essential genes. We report here asimple procedure to generate ts mutations using error-prone PCR within pcp1 that encodes aspindle pole body (SPB) component in Schizosaccharomyces pombe. This manipulation is not restricted to pcp1, and can be suited to any essential genes involved in other processes.
Highlights
When we wish to create ts mutants in particular genes, the implementation of error-prone polymerase chain reaction (PCR) becomes instrumental; with this methodology, we can generate desired ts mutations in GOI and efficiently
The procedure for isolation of ts mutants in S. pombe is schematically shown in aCC-BY 4.0 International license
It is important to use the genomic DNA derived from the strain carrying a kanR marker insertion in the 3’ end of the pcp[1] gene as a template
Summary
When we wish to create ts mutants in particular genes (gene of interest, GOI), the implementation of error-prone polymerase chain reaction (PCR) becomes instrumental; with this methodology, we can generate desired ts mutations in GOI and efficiently. We describe screening for ts mutants using this error-prone PCR method in the pcp[1] gene, which encodes the vertebrate The procedure for isolation of ts mutants in S. pombe is schematically shown in aCC-BY 4.0 International license. We performed random mutagenesis by error-prone PCR to introduce mutations within the pcp[1] gene (Fig. 1, II).
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