stability/storage of biotinylated primers (for PCR enrichment)

[pyridine]

Is anyone aware of the stability and storage recommendations for 5'-biotin-TEG modified oligos?

I'm using a rather expensive biotinylated primer during the PCR enrichment step of a DNA library preparation for later affinity purification using streptavidin beads, but the amplification efficiency has dropped 5-10X from the previous time it was used. Not knowing what else to do with it, I had resuspended the primer originally to 10 uM in water and it's been freeze-thawed a few times. This has never given me the slightest problem with any regular oligo, but I've seen precipitation in free biotin solutions that have been freeze-thawed so it looks like a really bad idea in retrospect...

[Genohub]

This is a fairly stable modification. I recommend aliquoting your reconstituted oligo into reaction size volumes and storing at -20 celsius in a non frost-free freezer. The aliquots should be stable for at least 6 months.

Are you trying to read through the biotin during PCR?

- Genohub

[austinso]

Oligos should always be resuspended in TE pH 8.0, or at least 1/10 TE.

You could spike in a 10x solution of TE to preserve what you have, but at this point I would probably get them remade.

[pyridine]

I was trying to avoid adding EDTA because I don't want it to inhibit the PCR reaction at all. Does the amount of EDTA in TE not usually cause problems? I suppose I've used genomic DNA in TE without problems as a PCR template in the past, but I'm using a somewhat higher volume of the primer.

Since the biotin is on the 5' end, it wouldn't be reading through the biotin but I am using it to do a PCR enrichment.

I'll have it remade and will aliquot it which should eliminate most problems, but still not entirely certain what to dissolve it in. But at least 10 mM Tris pH 8 this time around.

Thanks for the replies.

[austinso]

I was trying to avoid adding EDTA because I don't want it to inhibit the PCR reaction at all. Does the amount of EDTA in TE not usually cause problems? I suppose I've used genomic DNA in TE without problems as a PCR template in the past, but I'm using a somewhat higher volume of the primer.

Since the biotin is on the 5' end, it wouldn't be reading through the biotin but I am using it to do a PCR enrichment.

I'll have it remade and will aliquot it which should eliminate most problems, but still not entirely certain what to dissolve it in. But at least 10 mM Tris pH 8 this time around.

Thanks for the replies.

10 mM Tris pH 8 will work fine (I guess I should have specified that a buffered solution is important, not necessarily the presence of EDTA). And avoid freeze-thawing is key.

Generally speaking the amount of Mg2+ is 2-4 mM in a PCR reaction (depending on the DNAP system used). Adding up to 1/5 volume of a TE will not generally impact the reaction (~0.2 mM EDTA).

A.