Blocking Unspecific sites

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sdelandtsheer
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Blocking Unspecific sites

Post by sdelandtsheer » Thu Jan 26, 2006 4:22 pm

Hello,

I am actually doing an experiment consisting in fishing out some DNA with a biotinylated probe, using magnetic beads and streptavidin...

Does anybody knows how to block unspecific binding ? I plan to use calf thymus DNA, but I am not sure about the excess to use, and maybe BSA would also block unspecific DNA binding to the beads....

An idea ?

Thank you all in advance

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NKCC
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Re: Blocking Unspecific sites

Post by NKCC » Thu Jan 26, 2006 10:22 pm

sdelandtsheer wrote:Hello,

I am actually doing an experiment consisting in fishing out some DNA with a biotinylated probe, using magnetic beads and streptavidin...

Does anybody knows how to block unspecific binding ? I plan to use calf thymus DNA, but I am not sure about the excess to use, and maybe BSA would also block unspecific DNA binding to the beads....

An idea ?

Thank you all in advance


I don't have a lot of experience in the DNA area, but as far as antibodies, radioisotopes, and even protiens are conserened, to block non specific binding, we usually use excess protein or blocking agent. In your case, if you are worried about random DNA segments bindign to the beads, then treat the beads with a know solution that contains a know DNA segment. In this case, you will know excatly what your noise is. Another thing is that, since the binding site of the beads bind to a specific segment of DNA with some affinity and nonspecific DNA bind to other parts of the bead with lesser affinity, you can heat the beads at the end and disassemble the nonspecific DNA. Be careful because heating too much takes everything off of the beads including what you are trying to isolate. So, this technique requires seperation of the beads using magent twice, once after exposure to the cell lysate and once after the heat treatment.

Just an idea.

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canalon
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Re: Blocking Unspecific sites

Post by canalon » Thu Jan 26, 2006 10:37 pm

sdelandtsheer wrote:Hello,

I am actually doing an experiment consisting in fishing out some DNA with a biotinylated probe, using magnetic beads and streptavidin...

Does anybody knows how to block unspecific binding ? I plan to use calf thymus DNA, but I am not sure about the excess to use, and maybe BSA would also block unspecific DNA binding to the beads....

An idea ?

Thank you all in advance


I am not familiar with your technique but I would use DNA. The quantity of excess would probably a few times the maximal quantity of DNA that can be bound. And check that the blocking reagent do not cross react with the following steps of your reaction.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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Dr.Stein
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Post by Dr.Stein » Fri Jan 27, 2006 7:23 am

I am new to this technique, I have no idea :oops:
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