DNA-Bead Attachment Protocol: Streptavidin & Biotin/DIG

  • Thread starter karthik3k
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    Bead Dna
In summary, the commercial beads come with a protocol for DNA-streptavidin attachment. You can mix the beads with the DNA at room temperature for 10-15 minutes, and the conditions for SA/Biotin and DIG/Anti-DIG binding should be similar.
  • #1
karthik3k
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Does anybody have a good protocol for DNA - bead attachment ?
The Bead is streptavidin coated and the DNA has biotin and DIG on its ends.
 
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  • #2
Streptavidin will bind directly to biotin. I haven't done this particular procedure, but the streptavidin-biotin binding should occur at room temperature (I don't know how much of a range outside of room temp you can use). If you know how many biotin binding sites you have on each strand of DNA, you can determine the amount of streptavidin beads to add and hopefully just mix. Where did you purchase the streptavidin coated beads from? Do they have a recommended protocol?
 
  • #3
We don't buy readymade strep coated beads. We make them...

Do u have any published protocol for DNA-Bead attchment?
 
  • #4
You make them yourself, but don't know how to use them?

Anyway, this isn't something I work with, so you could look up the information just as well as I can. Here's one commercial supplier with a protocol for their product on this page: http://www.dynalbiotech.com/kunder/dynal/DynalPub401.nsf/$all/841E0473B0D7A8FAC1256C5400488DF4

It looks pretty straightforward.

A google search using the keywords streptavidin, beads, and DNA comes up with quite a few hits. You could compare what different manufacturers recommend and see which fits best with your own beads.

You could look for other published protocols on PubMed:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi
 
  • #5
hey thanks :)
but DynaBeads catalog talks only about DNA-Biotin Bead attachment.
But what about DNA-DIG AntiDIG attachment ?

I didnt get any papers where the give out the actual protocol...
 
  • #6
There are commercial beads which come coated w/ SA or Anti-Dig. If you're talking about the buffer conditions needed for SA/Biotin attachment, it should work in your normal DNA buffer (ie pH 7 buffered, NaCl). As previously mentioned you can just mix the beads with the DNA at RT for 10-15 min. Should be plenty of time. Dig/Anti-Dig although a weaker interaction than SA/Biotin will work pretty much under similar conditions.
 
  • #7
Actually the problem is ...
I want to attach one end of the DNA to SA coated bead. and other end to the anti-DIG coated glass slide.

Now the problem is i want to know the conditions for DIG , anti-DIG binding...
Can i use TE with Nacl (1X) buffer for this ??

What about the pH conditions ??

Does NaN3 degrade anti-DIG ? if not how does it react ??
somebody please help ...
 
  • #8
I have worked with anti-dig for EMSA and the buffer for detection was:

1% Blocking reagent (w/w) in
Maleic acid buffer (100 mM Maleic acid, 150 mM
NaCl, pH 7.5).

http://www.roche-applied-science.com/pack-insert/1093274a.pdf [Broken]
 
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What is the purpose of the DNA-bead attachment protocol using Streptavidin & Biotin/DIG?

The purpose of this protocol is to attach DNA molecules to beads through the use of Streptavidin and Biotin/DIG. This allows for the isolation and purification of specific DNA sequences for further analysis.

What is Streptavidin and how does it work in this protocol?

Streptavidin is a protein that binds to biotin with high affinity. In this protocol, Streptavidin is used to bind to DNA molecules that have been biotinylated, allowing for the attachment of the DNA to the beads.

Why is biotinylation of DNA important in this protocol?

Biotinylation of DNA is important because it allows for the specific attachment of Streptavidin to the DNA molecules. This ensures that only the desired DNA sequences are isolated and purified.

What is the role of DIG in this protocol?

DIG (Digoxigenin) is a small molecule that can be covalently attached to DNA. In this protocol, DIG is used in place of biotin to label the DNA molecules. This allows for flexibility in the attachment process and can be used in combination with biotinylated DNA.

What are some potential applications of the DNA-bead attachment protocol using Streptavidin & Biotin/DIG?

This protocol has many applications in molecular biology, including DNA sequencing, DNA-protein interaction studies, and isolation of specific DNA sequences for cloning or further analysis. It can also be used in diagnostic tests for genetic disorders and forensic analysis.

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