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I'd like to produce a specific DNA sequence on a milligram-scale and 13C15N-label it. The sequence is around 35 nucleotides long, so chemical synthesis is out due to the exorbitant costs.

I'm also only interested in the single-stranded DNA, so a method that produces double-stranded DNA without any way to easily separate the strands would also not be useful.

What methods are there to produce labelled DNA that fulfill these requirements?

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4 Answers

You can also design a plasmid that has your 35mers and raise the bacteria with isotopic carbon and nitrogen C13 acetate and N15 ammonium sulfate will work with E coli in a minimal medium.

this is not cheap, but the cheapest available sources of the isotopes can be used.

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That doesn't give me single-stranded DNA. It's also very inefficient as I only use 1/200 of the plasmid DNA (30bp single-strand of a 3kb plasmid), so a Megaprep with 2.5 mg yield would still only give me 0.0125 mg of my DNA. I can't reasonably make 100 Megapreps to isolate 1mg of my sample. –  Mad Scientist Dec 15 '11 at 20:49
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I found a couple of methods using M13 phage growing on E coli:

You could easily adapt these protocols to grow on 13C, 15N-containing medium. Then you could use a restriction enzyme able to cut ss DNA and purify your fragment through a PA gel. Clontech's gigaprep kit (up to 50 mg of DNA) will cost you $546 (in the US, before any quote).

Disclaimer: I've never done any of this!

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The cheapest way to make that large quantity of DNA without the need of the whole oligomer being label would be to produce 34 nucleotides by chemical synthesis. The 35 nucleotide can be labeled and ligated to the remaining strands. This should reduce the costs of producing the oligomer since the most expensive stage, the labeling is limited to just the final nucleotide.

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I need all of the nucleotides labeled, only the last one is not enough unfortunately. –  Mad Scientist Jan 22 '12 at 10:01
    
I realized that the other suggested ideas are pretty effect. I would culture the plasmids with non-labeled bacteria and using a cesium gradient, you should be able to separate half-labeled DNA from unlabeled and fully labeled DNA. After a restriction digest, you should be able to isolate single stranded DNA by density centrifugation. –  bobthejoe Jan 23 '12 at 6:32
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What about PCR using labeled nucleotides? Might have to run several reactions to get miligram quantities you need, but 35 nucleotides seems really really small for growth in bacteria, and purification would be extremely difficult. But 35 bases might even be too small for PCR, hardly bigger than your primers.

If you do need to grow in e coli, it might make sense to create a plasmid with multiple repeats of the 35 base sequence separated by the same restriction site. You can grow e coli in c13 n15 media, but it's expensive and you have "train" the cells over several generations because the heavier isotopes create differences in chemical kinetics that make the labeled nutrients hard for enzymes to use, but I've seen people do this for making proteins for NMR.

I bet you need this labeled DNA for NMR, don't you?

Have you considered phosphate NMR? No need to label it, P-31 is already NMR active. If it must be C13 N15, I highly recommend having it synthesized, it will be expensive, but that 35 base length and the requirement for single strandedness are ideal for chemical synthesis.

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