Computational protocol: Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies

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Protocol publication

[…] For the initial check of each isolated plasmid, ~500 ng of the plasmid DNA was taken for sequencing. We have a set of primers that we routinely use for sequencing ENaC cDNAs. For each mutation we selected a sequencing primer about 50–300 bp away from the point of mutation. Sequencing reaction was carried out in a final volume of 20 μl, with the addition of 20 pmol primer, 3 μl sequencing buffer and 2 μl of BigDye® Terminator (ABI). PCR for sequencing was carried out following ABI protocol: Initial denaturation step at 96°C for 1 min followed by 25 cycles of PCR with denaturation at 96°C for 10 s, primer annealing at 55°C for 5 s and extension at 60°C for 4 min. The PCR product was then analyzed using an ABI 310 Genetic Analyzer. If the sequence of the mutated region was found as expected, then the whole cDNA insert was sequenced to ascertain that the cDNA sequence does not carry any other mutation.The sequence files in ab1 format were analyzed using FinchTV software (Geospiza Inc.). Sequence alignments were done using BioEdit []. […]

Pipeline specifications

Software tools FinchTV, BioEdit
Application Sanger sequencing
Organisms Escherichia coli, Homo sapiens
Chemicals Nucleotides