Biotinylated oligo separation - (Aug/02/2010 )

Hi,

I need to separate biotinylated oligos (21mer) from my reaction which contains larger DNA (>3kb). Would a centrifugal filter or a PCR purification kit do the job? Also, suggestions about specific products/brands would be much appreciated. Thanks!

mray on Mon Aug 2 17:32:59 2010 said:

why not using streptavidin coated magnetic beads and then a strong magnet? Dynabeads from Invitrogen is one brand (though expensive)

Hi,

I separate Biotin-labeled 23mer using Dynabeads from Invitrogen. It always worked fine and the recovery is quite good. May be you can give a try.

hobglobin on Mon Aug 2 18:53:54 2010 said:

I guess I didn't explain myself very well. In my experiment, the biotinylated oligo binds to a larger piece of DNA which is what I want to selectively isolate using magnetic separation (Dynal kilobase binder kit). But I need to get rid of excess oligo or it competes with the target DNA for binding sites on the streptavidin coated magnetic beads. Hope that makes more sense

HI...
I think my suggestion can help you....
why not using streptavidin coated magnetic beads and then a strong magnet? Dynabeads from Invitrogen is one brand (though expensive)...
try????
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mray on Mon Aug 2 23:03:10 2010 said:

Hi there
If I understand you correctly, you are doing a sequence specific DNA capture using Dynal kilobaseBINDER kit. This kit is developed for isolation of large, biotinylated DNA fragments. It contains Dynabeads M-280 Streptavidin and a special buffer to optimize capture of large, biotinylated DNA fragments. For binding of small oligos like you are using, this kit will not improve the binding efficiency, so it is a cheaper option to use Dynabeads M-280 Streptavidin, cat. no 11205D.

For sequence specific capture we recommend using Dynabeads M-270 Streptavidin as these beads are slightly negatively charged, resulting in very low background binding of DNA to the beads. Dynaebads M-280 Streptavidin have been used successfully for this application, but normally require more optimisation, and background binding can be a problem. We have a web-page about sequence specific capture of DNA using Dynabeads which yo might find helpful:
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Nucleic-Acid-Purification-and-Analysis/napamisc/Capture-of-Biotinylated-Targets/Sequence-Specific-Capture.html

This page also contains some references which might be helpful to you.

I recommend doing direct capture, i.e. binding the biotinylated oligo to the Dynaebads Streptaivdin first, then capturing the target DNA sequence.
To remove excess oligo, simply wash the beads after binding the oligo a few times using the magnet.

best wishes

Kristina