I need to delete up to 30 bp from a plasmid to generate deletion mutation. Has anyone tried using Agilent direct mutagenesis to delete longer regions? How feasible is this?
There is a modified method proved efficiently https://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91. You can delete/insert whatever you want easily.
We have successfully used the standard Quikchange II SDM kit to delete (and replace) about 30 residues (6xHis + linkers). You can do everything pretty much identically, just need to sequence more colonies as it has a lower success rate. Also, you will want to monitor the GC content of your primers more closely to avoid possible secondary structure forming in the longer primers.