Fusion PCR problem: smear! - (Oct/22/2007 )

I perform fusion PCR (2nd round PCR) with 2 outer primer and template from round 1. Why I got smear bands?. Although, I can see the upper band that is fusion PCR product that I'm expected to, I still see another band (the lower band) that is seem to be the template from round 1.

Someone told me to cut only upper band and amplify again. But, when I cut only the upper band to do the PCR in third round, the problem still occur.

How can I improve my PCR?

I use Taq polymerase in PCR, however when I use proofreading enz. (deep Vent) there is no product from the amplification.

Can anyone help me?

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how big is your product? If you don't mind, could you list your PCR conditions? Sometimes lowering the extention temperature can help promote production of the longer PCR product. Drop the extention temperature to 68 Celsius and increase your extention time by 40% to compensate. See if that helps.

And finally, how are you cloning in your PCR product? Are you yeilds good enough so that you can gel purify your desire bands. Remember the goal is to get your PCR product into a plasmid, not make a perfect reaction. As long as you can get sufficient PCR fusion product to clone that is good enough.

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Thanks for your advise! I'd like to fuse 617+640 bp together.
Each template has 19 bp overlaped.

This is my PCR conditions:

95 3 min

95 45s
57 45s 30 cycles
72 3 min

72 5 min

The problem is I haven't got a good yield of 1200 bp band, and also that band is interrupt by smear.

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The annealing T of this step should be higher than the last step, and I recommend you use a little longer overlap region, for instance your final product length is 1.2kb, the overlap: between 20 and 25. Do not use Vent polymerase during overlap extension PCR, it can not work. And the other importance is the primers used for the 2nd step should be estimated to make sure no false priming.

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