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Old 09-24-2012, 05:41 AM   #6
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Originally Posted by thdybwf View Post
But weirdly, when I analyze the template and PCR product with a Nanodrop, I get the following:

Nucl. Acid Concentration: 11.7 ng/ul
A260: 0.235
A280: 0.075
260/280: 3.15
260/230: 3.09

Nucl. Acid Concentration: 720.2 ng/ul
A260: 14.405
A280: 9.906
260/280: 1.45
260/230: 0.61

There seems to be a decent amount of DNA in both, and it's much greater in the PCR.
UV spectrophotometry is often misleading. For the reasons mentioned by others in this thread and a bunch of other ones, including some I detail here.

The nanodrop is easy to use, and the UV spectrum it plots can give you useful information -- if you bother to look at it. But whenever you perform an assay, you want to know its limits of sensitivity as well as anything that might confound it reading only (or mostly) what you are interested in (DNA concentration).

ingrid@illumina gives good about as good advice as can be packed into a couple of sentences. However Illumina (along with many other companies) does its level best to "black box" its protocols. That is, they tend to hide salient details in a misguided attempt to prevent others from reverse-engineering their protocols. That puts them firmly in the "part of the problem" camp in my opinion.

But, hey, I can't entirely blame Illumina because nobody seems to have taught you the basics of what a UV spectrophotometer detects. To a first approximation the answer is "lots of stuff", including DNA and RNA, but also nucleotides, acetic acid or salts thereof, salts of guanidium, phenol, DTT, etc. etc. etc. Don't feel too bad about it personally, more than 90% of bench scientists in molecular biology I meet are in the same boat. (Well, I am at a University, so these scientists are largely students...)

Then there is the issue of sensitivity. Can you detect a 10 nM library on a nanodrop? Worthwhile to keep a couple of "rules of thumb" in mind as you navigate a protocol. Some details here. But to a first approximation:

1 ul of a 1 nM solution contains 1 billion molecules (2nd approx.: ~60% of that.)

1 ng of a 1 kbp fragment is 1 billion molecules (2nd approx.: ~93% of that.)

Therefore if your library is 10 nM, that would be about 6 billion molecules/ul. If these molecules are 1 kbp, then that is about 6 ng/ul. But they are probably closer to 1/2 that length. So 3 ng/ul.

Can you reliably detect DNA at 3 ng/ul on a nanodrop? Not on ours. Below 10 ng/ul, things get pretty sketchy. How about on an agarose gel? Too many variables to say for sure, but probably not unless your library is in a very tight size range or you have a very sensitive system set up.

Hence the wisdom embedded in "ingrid@illuminas" advice -- go with an Agilent Bioanalyzer to QC your library. Using a high sensitivity chip you are good down into the pg range. Or fluorimetry/qPCR -- except you don't see the size of your products that way.

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