Overlap PCR problem - (Feb/16/2013 )

I am attempting to change 18bp in a 10.5kb plasmid. I have designed four primers for the overlap PCR. I have an overlap region of 18bp (the actual sequence I am attempting to change) in these primers.

The primers give two products at expected sizes and the overlap PCR yielded a product at the correct size (piece 1 + piece 2)

My problem is after I digest my vector and overlap PCR product and then ligate is all of my colonies have the wild type sequence. I have used overlap PCR with this same strategy before with no problems. Is it possible to amplify the WT sequence with mutating primers? The only thing I can think of is the digest of my vector was incomplete (1 bad enzyme) and I am just sequencing colonies that self-ligated. I might do a sequential digestion instead of a double digest to test this theory.

Has anyone had any experience with this type of issue. I can dive into specifics of my experiments if needed.

Thanks

last time I did deletion-PCR to remove nearly 10 bp from a 7-8 kb plasmid. it worked in the first run. I designed my primers to amplify backwards/opposite direction of the 10 bp location. before running on the gel I added DpnI to digest the template plasmid. then I transformed into competent cells to both grow more plasmid and fill the nicked plasmid

This is the original paper:
http://www.sciencedirect.com/science/article/pii/S0003269707007919

why do you have to do overlap-pcr for 10 k plasmid? isn't that more difficult? I used pfx polymerase from invitrogen.

I am not deleting a sequence, but changing 18bp to a different sequence. The overlap is the 18bp I am changing. So the primers are introducing the altered sequence. I opted for this instead of multiple site-directed mutagenesis reactions.

The first PCR I generate two products that have the altered sequence at the overlapping ends. The second PCR I mix the products and amplify the whole product that joins piece #1 and piece #2 with the changed sequence in the middle.

Curtis, I think the DpnI digestion could be used to prevent parental DNA from contaminating the overlap PCR where I would not be able to differentiate WT from changed. So I think I will treat my first PCR reactions with DpnI to prevent any carryover.

oh, I thought you want to delete it, sorry, I didn't read very well, but I think you can still use the same approach if your own method doesn't work. How long is the overlap region? I use about 21 bp usually. longer than that reduces my product.

Curtis, I got to the bottom of this. I did another overlap PCR using the same strategy, but I digested the PCR product with different restriction enzymes and then ligated it into my vector. 5/5 colonies were the correct sequence. The PCR I was having trouble with was due to a bad XbaI restriction enzyme and my vector and PCR product were only being cut once, thus all of the self-ligation and WT sequences. Really frustrating to spend the better part of a month on something that was doomed from the start.

congrats, you are lucky, it's 9 months we are struggling with our OE-PCR, we could have a baby by now.