Overlapping PCR, need help - (Sep/30/2014 )

Hi, I have been attempting overlap mutagenesis PCR with  2000 bp and 3000 bp fragments. They have a 27 bp overlap region. Both fragments were gel purified prior to overlap PCR. Here are te conditions.

5X buffer: 5ul

Primer F: 1.5ul 

Primer R: 1.5ul 

dNTPs: 1ul (10mM stock)

PCR fragment (each): 2.5uL 

Phusion polymerase: .2uL

dH2O to 25uL

PCR cycles

98C - 15s

50C - 20s            x35

72C- 5 min.

I either get smear or nothing at all. Most of the times I can see the 2000bp fragment, but I`ve noticed that the 3000bp fragment 

never appears. Also, I get a poor yield of the 3000bp fragment during the first PCR, contrary to the 2000bp fragment.

Can anyone help me?

You should verify that both fragments are really present. Run a gel and check that both the 2000 and 3000 bp fragments are there.

Make sure that you use very small amounts of both fragments as templates. Excess template is often a problem in these PCR reactions. Try using 10x serial dilutions of your template.

Hi Phage434, thanks for replying.

I'm sure both fragments are present before the PCR, however, it's as if the 3000bp degrades during the PCR, since it stops being visible afterwards.

How much template do you recommend? Enough for it to be seen in the gel or it shouldn't be visible?

It definitely should not be visible on a gel. Less than 1 ng, perhaps far less. Try a 100x or 1000x dilution from what you have tried.

Ok thanks, I'll definitely try it.

One more thing, do you recommend including the external primers since the beginning or after 10 cycles only with the templates?

I would add the primers in the beginnning, but I can see the reasoning to delay. If you delay, I would extend the number of cycles.

Would you be more clear about your cycling conditions. What exactly happens in each cycle? The way you have written it is not clear.