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Hello and thanks for reading.
I recently ran into a confusing issue with deletion mutagenesis. The approach I took to the deletion was to amply the entire plasmid, excluding the region to be deleted using 5' phosphorylated primers. PCR was followed by Dpn1 digest and ligation with T4 ligase. I transformed the PCR product into DH5a cells (cloning line) and prepared plasmid from individual colonies.
Sequencing shows that the deletion did work, however there is an additional base where the primers were ligated.
Specifically, the coding strand sequence should have read: ACC.CCT (ligation point is at the period)
and instead I got: ACCCCCT
Where the extra base causes a frameshift in the gene.
I should mention that all 3 colonies that had the deletion also had the extra cytosine.
Does anyone know what is going on here?
--Chris
