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Old 12-02-2015, 07:11 AM   #21
SunPenguin
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Is this a typo? Even starting from a single molecule, 70 PCR cycles would give you about 21 g of DNA, according to a quick back-of-the-envelope calculation, though of course you'll exhaust the reagents first (and primers are usually the first thing to go, by design). I'll grant you some loss in the intermediate steps, but still. Overamplification definitely sounds like a possibility.
hmm no it's not a typo, unfortunately. We started with very high cycle numbers and more rounds of PCR, in an attempt to make sure we're getting the right amplification. 70 cycles is divided in 3 steps, and only a small aliquot of product from each step is carried into the next. For gene amplification with nested PCR, I thought that's not really that unusual, though it may be a bit old fashion?

Phusion in our hands just gives the best amplification, so we've stuck with it. I've also tried Platinum Taq (in an effort to reduce chimeric product in exchange for higher error rate) and Kapa, though neither of those have worked as well. I haven't attempted to optimized those enzymes, however.
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Old 12-02-2015, 07:20 AM   #22
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The graph is consistent with my experience; 0.4X isn't going to work and even 0.5X is dodgy. For the record, you really should recalibrate your SPRI conditions for your specific reaction mix, because certain components (like magnesium, or especially PEG from a "quick ligation") change the binding chemistry.

But then your Bioanalyzer trace looks perfect except for being overamplified.

Instead of a denaturing gel, you can also denature a sample yourself (a couple of minutes at 95 C) and then run it on a Bioanalyzer RNA chip to profile the ssDNA. This won't get artifacts from daisy chains, bubbles, or whatever overamplification causes.
I see. I'll definitely look into recalibrating the SPRI.

After you denature the DNA at 95, do you then just let it cool back down to roomtempt? or do you have to try to run it as soon as possible?
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Old 12-02-2015, 07:22 AM   #23
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After you denature the DNA at 95, do you then just let it cool back down to roomtempt? or do you have to try to run it as soon as possible?
I've always just run it immediately, so I don't know what happens if you wait.
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Old 12-02-2015, 11:28 AM   #24
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I just ran a titrating PCR like you said, and ran a quick E-gel on it. It does seem reducing the template and cycle number concentrate the product more and produce less smear! though even just a few cycles too low, the product band decrease dramatically in intensity.

I'll put it through spri and run it on the BioA and update.
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