Hi Suma!

It is a bit strange and I am not sure to understand what you do mean when you say that you don't enrich. How do you check for binding?

Hi Abdelhalim,

I did two experiments:

1. I just took non-biotinylated DNA and incubated with magnetic beads. After incubation, I did PCR using beads as template and got amplification of the DNA I started with.

2. I had a mix of biotinylated and nonbiotinylated DNA. Each piece contains a specific restriction enzyme site. I incubated the mix with magnetic beads and did PCR using beads as template. I digested the amplified product with both enzymes and found that the non biotinylated DNA is also being amplified.

Therefore, I am not able to enrich the biotinylated DNA in my experiments as the beads are also capturing non-biotinylated DNA.

Thoughts?

Hi Suma,

Do you perform several washes before the PCR? As you know, PCR is very sensitive even if you initially enrich, after amplification you can end with same amount from both samples. So, unless you perform a qPCR you can't really tell. If this is the case then I am not sure what makes the issue but I suggest the following:

You could bring your sample to 2M NaCl salt before incubating with streptavidin magnetic beads and then perform several washes with buffer compatible for downstream application = to make it ready either for amplification or for restriction digestion. Let me know if it works for you

Hi Suma, I know it is an old thread, but have you resolved your problem? I am seeing the same thing as well. I have a biotinylated fragment bound to the M280 beads, and am using a competition reaction with unbiotinylated fragments of different sequences. What I see is, the unbiotinylated fragments are sticking to the beads pretty well. Even an initial blocking with salmon sperm DNA did not help. Abdelhalim: I am aware that the recommended DNA binding-wash buffer contains 2M NaCl. But people forget that these beads are supposed to be used in a wide range of experiments where you simply cannot exceed the physiological salt concentration. The best way here is to block non-specific DNA binding. Jon: I suppose people rule out silly contamination problems before they venture into a forum like this. I shall appreciate a constructive suggestion instead!

Hi Anil,

Yes. I solved the problem. Two things resolved the problem.

1. Keeping the buffer cold (I store my buffers at 4 C).

2. Rotate the tubes for 5 min at RT between washes.

Here is the exact protocol I used.

Take 10-20 ul M-280 streptavidin beads from invitrogen. Remove the buffer and wash the beads three times with 500 ul of 1X Tween buffer (5 mM Tris HCl pH 8, 0.5 mM EDTA, 1M NaCl, 0.05% tween).

Remove the buffer and add 20-40 ul (twice the volume of initial beads) of 2X No Tween Buffer ( 10 mM Tris HCl pH 8, 1 mM EDTA, 2M NaCl). Add 20-40 ul of sample DNA and incubate for 30 min with rotation at RT.

Remove the buffer and wash the beads two times with 500 ul of 1X Tween buffer and once with 500 ml of 1X No Tween Buffer.

Remove the buffer and resuspend the beads in 15 ul 1X TE. You can now use 1-2 ul of this suspension for PCR.

For my experiments, I used 20 cycles PCR and I didn't see any non-biotinylated DNA amplifying.

I would recommend you to do this procedure using non-biotinlated DNA only and see if you are getting any amplification.

Hope this helps! Let me know if this solves your problem.

Good luck!

Hi Suma, thanks for the detailed response. The procedures that you are following make perfect sense. Unfortunately for me, I have to maintain as mild a wash condition as possible--so that I do not disrupt any DNA-protein interaction. That means, I cannot go beyond 150mM NaCl and a milder detergent concn of, say, 0.1%. So, I guess the solution for me is to optimize the blocking such that unbiotinylated DNA does not stick to the beads. But thanks again for sharing your protocol.

I see. No problem. Good luck!

Hi Suma and Anil, this is an old thread but I had some question. I am starting new with the bead binding assays. I am trying to purify biotinylated DNA out of the restriction digestion reaction/ligation reactions. I can not heat inactivate the enzyme because the daptors I am ligating are short and heat inactivation will denature the DNA which will be a problem in my downstream processing. But if I continue with the bead binding incubation in 1mM EDTA, 10mM Tris, 1M NaCl, the restriction enzyme might continue to cut as well. So I am planning to do the bead binding in 20 mM EDTA, 10mM Tris HCl and 1M NaCl. Do you think it will cause a trouble at some point?. Thanks in advance...

Hi Sanjay, I have not done DNA binding experiment with that high EDTA conc., but you should be fine. I do not see the possibility of EDTA impairing biotin-streptavidin interaction. High salt concn is usually advised for biot.DNA-streptavidin binding, so 1M NaCl will be just fine. In my hand, though, there is still good binding of unbiotinylated DNA to streptavidin beads (agarose beads, M280 as well as streptavidin-coated plates), in common buffers in presence of up to 1mg/ml BSA (and I cannot use high salt). I hope that is not too critical to your experiment.

Thanks Anil. I will try it and see what I get in the end.

I don’t like shy people who hide because they aren’t sure about what they are thinking...They aren’t sure even when they know they are right. At least I have successfully done that..still doing it, though. So, it pisses me off that someone just disagrees with that without providing any input that might be very useful to all. So, it is not about voting down or up it is about sharing information and that what all RG platform is about. I wish that this shy person could unveil himself and instruct us what was wrong in my statement.

That should be simple and straight-forward: do the PCR, put your reaction on magnetic platform, take the supernatant, process as you desire (PCR-clean-up or gel isolation or ligation). Your PCR product should not stick to the beads--well, not all. Your beads will be too little in amount to trap the PCR product. But do not use too much of beads in PCR. If I start with 10ul beads and bind 0.25 pmoles of DNA, I serially dilute the beads to 1:2500 and then take 1ul as template for a 20ul PCR rxn. Hope that helps.

Hi Suma, just wanted to say thank you for the binding/wash step protocol! I think I need to increase the Tween, but otherwise you provided a great place to start!

Interesting side note: I did a 150mM-1M NaCl gradient wash step, and 1M actually gave me more nonspecific pulldown than 150mM and 600mM NaCl buffer. I'm thinking it's because the salt helps drive forward hydrophobic interactions (which is what causes nonspecific DNA-bead binding). So I'm going to try increasing the Tween to increase stringency.

Hi, Suma, have you solved the problem？I have the same problem,  I found that the beads are binding the target DNA. so I added some no target DNA to block the non-specific binding, but  it did not work. Could you give me some suggestion ?  thank you!

Hi, All, you are welcome to use NVIGEN streptavidin beads for DNA capture. Compared to other brand on market, our beads provide better DNA capture specificity that enabled better differentiation of patient vs. normal people sample for rare mutation detection.

http://www.nvigen.com/Sequence_capture.php

Transfering beads to a fresh tube for the last wash should help reduce the non-specific problem too. There is often some non-specific DNA binding to tubes that might get carried over.