Asked 10th Jun, 2022

Stability of functional groups on DNA oligos during long term storage and defrosting/refreezing?

Dear global colleagues,
Do you have insights into the stability of functional groups (I am particularly interested in phosphorylation and Acrydite modifications)? IDT, for example, has detailed oligo storage advice on their website and it seems that olives are quite stable at -20 for up to two years and even a few defrosting cycles. However, they do not give details for functionalisation and I know from my own experience that biotinylation is quite sensitive (I estimate only a year storage is OK without any intermediate defrosting). What about the other functional groups? Wikipedia suggests Acrydite is also less stable. How long would you use what kind of functionalisation?

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How does Ovation RNA seq V2 kit produce double stranded cDNA?
1 answer
  • Praveen SinghPraveen Singh
The Protocol and workflow of the SPIA amplification process show linear amplification of cDNA, which is single-stranded (As per the schematic provided by them). However, the kit claims it generates double-stranded cDNA, which is not shown in the figure or explained how it is done!!!
Can somebody please help me understand this?
How does the Ovation RNA seq v2 kit generate double-stranded cDNA instead of single-stranded cDNA??!!
Nugen/ tecan suggests using their library preparation kits (for Illumina) which can utilize only dsDNA templates but not single-stranded.
They seem to work well, but I don't understand how the dsDNA is generated in the amplification step (RNA Seq V2 kit).
Please help.

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A new "polystyrene biotin support" has been synthesized for the solid support synthesis of the 3'-biotinylated oligonucleotides. Several oligos were synthesized and were analyzed by the HPLC and Mass Spec. Oligo analysis revealed that the biotin gets oxidized to "biotin sulfoxide" during the synthesis.
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