Hello all,

I'm trying to do minigene assays for multiple genes. I got 5 genes to work for the wildtype using pET01 exon trap vector, and the splicing assay worked fine.

Now I'm trying to generate mutated plasmids. Most of my mutations are inside the introns of the cloned genes, so the regions are very repetitive (in general).

I used the Takara tool to design the primers, and used standard desalting. Then I did the PCR for my mutated plasmids using 1ng of the WT plasmid as input, and TM recommended by NEB at 30x cycling. I gel purified the products, then used KLD to ligate and digest the mutated plasmids (1-2ul of the purified product was used).

I transformed into NEB High Efficiency cells with 5ul of the KLD reaction mix. All transformations worked fine (not too many, not too little bacterial colonies) and I sequenced 3 colonies for each gene.

But I'm getting the same problem for all of my 5 genes. Instead of just having my mutation, I'm getting some mutations on my gene region and the main problem: instead of having the single base mutation that I need, all my genes have part of the primer inserted together with the mutation.

I checked and all my primers were designed with 8-9bp overlap, ~30nt lenght, as recommended by many articles.

My plasmids range from 5kb to 8kb

What could I do to eliminate this type of unwanted insertion?

Does Page purified gel helps? Or should I just sequence more colonies until I find the one with only my mutation?

I'm worried about sequencing too many colonies as 3 for all genes had the primer inserted.