Tuesday 14 June 2011

biochemistry - How can I produce milligram quantities of an isotope-labeled DNA oligomer?

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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