Oligo precipitation

Steve at genedetect.com Steve at genedetect.com
Fri Jul 12 00:28:45 EST 2002


Hi Alesia

Here is a protocol. This is a standard oligonucleotide precipitation protocol we use to clean our oligonucleotide probes.

Adjust volumes used below relative to your starting volume.

We start with 5ug oligonucleotide in 55ul.

1. Add 150ul precilled (-20C) 100% EtOH. Sigma molecular biology grade.
2. Add 8ul of 4M LiCL (Sigma Molecular Biology grade again, supplied at 8M).
3. MIX WELL. 
4. Leave at -20C to precipitate (for 2 hours)
5. Centrifuge at 4C, 16,000g for 20 mins.
6. Carefully remove supernatant without disturbing pellet.
7. Wash pellet with 200ul 70% ETOH (also pre-chilled at -20C)
8. MIX TO DISSOLVE PELLET
9. Centrifuge at 4C, 16,000g for 20 mins to reform pellet
10. Carefully remove supernatant without disturbing pellet.
11. Dry pellet under vacuum for 2.5 to 3 hrs (i.e. with Eppendorf concentrator 5301)

Thats it.

Regards




> I wonder if anyone can help me undo a mistake.  I mistakenly
> diluted modified oligos in 20uM Tris buffer down to 10uM
> instead of the concentrated 100uM stock I wanted.  I'm
> afraid the increase in Tris will affect my assay(virus load
> detection via RT-PCR).  Does anyone have a suggested
> protocol for concentrating oligo's by precipitation while
> eliminating the salts?
> 
> best regards,
> 
> Alesia
> 

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