Primer Optimiztion - can this be done in water (Nov/03/2006 )
I am gong to optimize 6 new primers. In order to save time and cDNA, I thought I would run the six primers in water only with primer concentrations of 400 nm, 300 nm, 200 nm, 100 nm, and 50 nm, and then look at the dissociation curves (ABI Prism 7900), before testing a smaller group of primers with cDNA. Does this sound like a good plan?
I would rather do it using a plasmide as template
what's the point of doing it with water? that will not show you what you need to see. I think you need some sort of template, preferably known concentration
But don't you get primer dimers in the absence of template? If that is what you are trying to determine, then I don't see much of a problem with this
yeah, but varying template concentration can change the amount of your product that is primer-dimer. you may see dimers that form with water as the template, that disappear or reduce to manageable amounts when you add an appropriate DNA template. so I would expect to see dimers from time to time if you use water; I would not expect those dimers to always be an issue when template is present
but I'm not an expert I have done plenty of PCR, but there are others here who are really gurus that might disagree?
But if you have dimers at low target copy numbers, then you will not have a linear standard curve and thus the reaction will not be perfect for quantiitation.
I would still be reluctant to use water
you need to know how the primers will perform 'in the real world'
Thanks to all for your input. Here is a little follow-up:
I tested all of the primers with water, and then chose the three concentrations with the least amount of primer dimer to test with the cDNA. The quantities of starting material are really limited so I was able to save some cDNA for actual testing rather than using more of it up optimizing primer concentration. Overall it worked very well.