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Hi, I cannot use NiCl2 and I am having trouble immobilizing DNA onto mica using MgCl2 buffer (10 mM HEPES, 10 mM MgCl2, PH 7.5). The stock DNA is diluted 40x in this deposition buffer. After depositing the DNA onto mica for 1 minute, it is rinsed with double distilled water and dried with Argon.
Most of the time I observe DNA that is clumped up forming spheres on the surface. Sometimes it works and I see clear linear DNA. It's very irreproducible.
Any suggestions? Did anyone encounter such problems? Many thanks for the tips! -Tamara
Hi, Tamara
It is not uncommon that even with passivation using divalent ionic solution DNA will aggregate on the surface. We believe that is a part of the drying process. We tried to remove the liquid gentlely and slowly to maximize the adsorption and minimizing aggregation. Sometimes it helps.
Another way to prepare for DNA imaging (that many have used) is to silanize the mica surface or coated with polylysine. The exact protocol can be found here: http://www.veeco.com/nanoscaleworld/media/p/662.aspx
The goals remain the same:
(1)To immobilze negatively charge DNA on to negatively charge mica by passivation of positive charges.
(2) If the aggregation persist, you may want to image your DNA in fluid under physiological conditions (with any of immobilization protocols mentioned above).
Hope this helps.
Best wishes for your research,
Sincerely,
James