Hello,
I am purifyning fluorescent oligos through HPLC. My desired fraction of the mixture elutes at ~36% acetonitrile in triethylammonium acetate buffer, pH 7.0. I want these fractions to be stored for long term. I had tried lyophilization, but on resuspension in NFW it didn't show any peak in HPLC. I had also tried ethanol precipitation of the fraction, but it gave very low yield. Can someone suggest the way I can process these fractions to store for longer with a better yield, as we get the synthetic oligos from commercial sources?
Thanks
Surbhi
Most commercial oligos are dried by centrifugal evaporation or by freeze drying. Typically they are purified by prep HPLC and then dried. I am therefore surprised that you need to purify further.
Mainly - damage occurs to the dye (bleaching) or to the linkage (cleavage).
If the dye bleaches then you will see nothing in your constuct, and protection of the sample(s) from sources of light are essential. Shielding the door on the lyophiliser or covering the flasks with aluminium foil can help.
Generally the same measures can help protect the linker from clevage, you will be able to identify clevage becuase in HPLC-MS you will see two peaks (dye and oligo separately) not one. You typically see only one peak with bleaching.
Links to some commercial drying papers for oligos can be found here:
https://www.spscientific.com/ContentBlock.aspx?id=3062
Frequently - many commercial oligo providers do not lyophilise due to the high static charge carried by DNA, the few ug then "fly" to the nearest static surface, a glove, pipette tip, anything but where you want it. Typically we see concentration to dryness such that there is a dry film. I suggest that you check to are getting the oligos back into suspension in buffer. The oligo provider should have resuspension guidelines.
Kind regards,
Rob
Thanks Rob. I want to process further, not purify. The information was helpful. I will try this centrifugal drying method.
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