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Old 06-07-2013, 03:15 AM   #1
SS00
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Location: La Jolla

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Question Streptavidin Beads Q - Save Instead of Discard Supernatent

Hi all,

I have a question about streptavidin beads. I am looking to separate ssDNA DNA strands with strepatavidin beads using biotiynlated antibodies specific to a certain base. The problem is, I want to keep the DNA that does NOT bind to the beads (i.e. doesn't contain the base specific for the biotinylated antibody).

This will be in my supernatant, which is usually discarded. How do I recover the DNA from this supernatant? I am not sure SPRI beads will work to recover DNA from the supernatant as they will be ssDNA (from snap-chilling).

I know with SPRI beads the bead buffer contains PEG which is the basis of its selective binding ability, so to recover the DNA from my supernatent I only need to add more SPRI beads in excess of a 1.8x ratio. Will this theory work with streptavidin beads or are there any other bead types that would be useful?

Any help will be appreciated
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Old 06-07-2013, 03:29 AM   #2
ETHANol
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SPRI beads (AMPure) work on any nucleic acid, dsDNA, ssDNA, RNA, DNA/RNA hybrid. The higher the PEG/NaCl concentration the smaller the fragments you recover, albeit the secondary structure of single stranded nucleic acids may have some influence on their cut off but if your molecules are big >200 bp I don't imagine any issues.
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Old 06-07-2013, 03:36 AM   #3
SS00
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Thanks for the quick reply. So do you think the following workflow would work?

1. Start with my DNA sample - some strands contain a specific base I do not want (e.g. lets say uracil)
2. Add biotinylated antibody specific for certain base (e.g. uracil)
3. Add streptavin beads to bind only DNA that is bound to the biotinylated antibody
4. Separate on magnetic stand
5. KEEP supernatant, discard tube with beads
6. Add SPRI beads to my supernatant
7. Incubate for 15 min, wash with EtOH and dry
8. Elute dried SPRI beads with resuspension buffer, incubate for 2 mins, separate and keep supernatant.

My supernatent should now hold my ssDNA without the base I selected against, correct?
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