Forum :: DNA and carry over problems


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This forum is intended for questions about kinetics, Surface Plasmon Resonance and the instruments related to these techniques.
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TOPIC: DNA and carry over problems

DNA and carry over problems 25 Apr 2017 19:24 #1

Hi all,

We have immobilized biotinilated DNA in a neutravidin chip. We have problems of carryover and after different experiments we don´t know how to solve it. Even we don´t know if it can be a problem in the fluidic system.

Anyone could help

Thanks

DNA and carry over problems 25 Apr 2017 20:09 #2

Hi Papillon,

You can test the carry-over by injecting a high salt solution followed by a flow buffer injection. In Biacore type of instruments there are several wash routines that you can use to wash needle and/or IFC between injections to minimize carry-over.

To test the fluidics: In a system with four channels start detection on all four channels and inject high salt (e.g. 0.5 M NaCl or the solution that gives you problems) over flow channel 1 followed by an injection with flow buffer. Then repeat for ch 2, 3 and four. You should only see the bulk salt response in one channel at the time. If the salt response appears in the other channels, the micro fluidics is leaking.

Kind regards
Arnoud

DNA and carry over problems 26 Apr 2017 10:22 #3

Hi Arnoud,

Thanks for the answer. However, I think I have not explained properly the problem. After the immobilization of the DNA we passed NaOH 2mM, as it is described in the literature, then the buffer PBS and we see these (attached document)





Thanks again,

Papillon
Last Edit: 26 Apr 2017 10:25 by papillon. Reason: add attachment

DNA and carry over problems 27 Apr 2017 10:58 #4

Hi again,

I have test the system as you have said and no problems of leaking. We think that there are some problem on the fluidic system. I means, perhaps something that could be stick.

Do you have recived the attached doccument that I previusly sent? If not please tell me how I can incorporate it.

Thanks again.

Papillon

DNA and carry over problems 27 Apr 2017 16:52 #5

Hi Papillon,

There was no attachement to the previous post. To know how to attache a file, please read the image entry at the top of the general discussion. This is mainly for image files not for datafiles.

To minimize sticking you could try to add some extra detergent (~0.02% P20), salt (250 mM NaCl) or BSA (up to 1 mg/ml) if this is compatible with your system.

Arnoud

DNA and carry over problems 27 Apr 2017 17:09 #6

Hi Arnoud,

Steps 4 immobilization of the DNA, 5-PBS, 6-NaOH 2mM and 7-PBS again


LigandchannelcDNA.png
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