DNA-Bead Attachment Protocol: Streptavidin & Biotin/DIG

[karthik3k]

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.

 

[Moonbear]

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?

 

[karthik3k]

We don't buy readymade strep coated beads. We make them...

Do u have any published protocol for DNA-Bead attchment?

 

[Moonbear]

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

 

[karthik3k]

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...

 

[noobie]

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.

 

[karthik3k]

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 ...

 

[iansmith]

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