HI, i am encountering some issues with the Q5 site directed mutagenesis kit from NEB. I am supposed to do something very simple in principle which is delete a region of ~100 nt from my template plasmid Which is around 13kb. I have run the pcr reaction as the kit suggests and run the product on a gel to check it was a single band of the desired size. I have then done the KLD reaction and the transformed the competent cells that are present in the kit. After transformation I got many colonies and i have then proceeded to minipreps. However all the resilt plasmid run very bad on gel showing a size of 3-4 on and containing only portions of my template, including the ampicilline resistance. When I talked to the support team from neb they suggested me to change competent cells to deal better with plasmid bigger than 10 kb. What do you think is happening? Do you think the competent cells are the problem? Thanks :)
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